Fusion proteins and combination vaccines comprising haemophilus influenzae Protein E and Pilin A

ABSTRACT

The present invention relates to compositions comprising Haemophilus influenzae Protein E and Pilin A. More particularly, the present application relates to fusion proteins and immunogenic compositions comprising Protein E and PilA, vaccines comprising such immunogenic compositions and therapeutic uses of the same.

This is a Divisional Application of pending U.S. application Ser. No.14/571,546, filed Dec. 16, 2014, which is a Divisional Application ofU.S. application Ser. No. 14/110,857, filed Oct. 9, 2013 (now U.S. Pat.No. 8,945,577, issued Feb. 3, 2015) which was filed pursuant to 35U.S.C. 371 as a U.S. National Phase Application of International PatentApplication Serial No. PCT/CA2012/050236, filed Apr. 12, 2012, whichclaims priority to U.S. patent application No. 61/534,012 filed Sep. 13,2011, and to U.S. patent application No. 61/474,779 filed Apr. 13, 2011,and the entire contents of each of the foregoing applications are herebyincorporated by reference.

FIELD OF THE INVENTION

The present invention relates to compositions comprising Haemophilusinfluenzae (H. influenzae) Protein E and Pilin A. More particularly, thepresent application relates to fusion proteins and immunogeniccompositions comprising Protein E and Pilin A, vaccines comprising suchimmunogenic compositions and therapeutic uses of the same.

BACKGROUND OF THE INVENTION

Protein E (PE) is an outer membrane lipoprotein with adhesiveproperties. It plays a role in the adhesion/invasion of non-typeableHaemophilus influenzae (NTHi) to epithelial cells. (J. Immunology 183:2593-2601 (2009); The Journal of Infectious Diseases 199:522-531 (2009),Microbes and Infection 10:87-96 (2008)). It is highly conserved in bothencapsulated Haemophilus influenzae and non-typeable H. influenzae andhas a conserved epithelial binding domain. (The Journal of InfectiousDiseases 201:414-419 (2010)). Thirteen different point mutations havebeen described in different Haemophilus species when compared withHaemophilus influenzae Rd as a reference strain. Its expression isobserved on both logarithmic growing and stationary phase bacteria.(WO2007/084053).

Protein E is also involved in human complement resistance throughbinding vitronectin. (Immunology 183: 2593-2601 (2009)). PE, by thebinding domain PKRYARSVRQ YKILNCANYH LTQVR (SEQ ID NO. 1, correspondingto amino acids 84-108 of SEC) ID NO. 4), binds vitronectin which is animportant inhibitor of the terminal complement pathway. (J. Immunology183:2593-2601 (2009)).

Pilin A (PilA) is likely the major pilin subunit of H. influenzae TypeIV Pilus (Tfp) involved in twitching motility (Infection and Immunity,73: 1635-1643 (2005)). NTHi PilA is a conserved adhesin expressed invivo. It has been shown to be involved in NTHi adherence, colonizationand biofilm formation. (Molecular Microbiology 65: 1288-1299 (2007)).

Non-typeable Haemophilus influenzae is an important and commonrespiratory pathogen that causes otitis media in infants and children.NTHi is, after Streptococcus pneumoniae, the most common cause of acuteotitis media in children (J. Immunology 183: 2593-2601 (2009),Pediatrics 113:1451-1465 (2004)). It is an important cause of sinusitisin children and adults. (Current Infectious Disease Reports 11:177-182(2009)). It has been associated with increased risk of exacerbations inchronic obstructive pulmonary disease (COPD) in adults. (Journal ofChronic Obstructive Pulmonary Disease 3:109-115 (2006)). In addition,non-typeable H. influenzae causes community-acquired pneumonia in adultsand may cause pneumonia in children in developing countries. (CurrentInfectious Disease Reports 11:177-182 (2009)).

A need for vaccines for NTHi exists.

BRIEF SUMMARY OF THE INVENTION

As a first aspect, the present invention provides fusion proteins offormula (I).(X)_(m)—(R₁)_(n)-A-(Y)₀—B—(Z)_(p)  (formula I)

wherein:

X is a signal peptide or MHHHHHH (SEQ ID NO. 2);

m is 0 or 1;

R₁ is an amino acid;

n is 0, 1, 2, 3, 4, 5 or 6;

A is Protein E from Haemophilus influenzae or an immunogenic fragmentthereof, or PilA from Haemophilus influenzae or an immunogenic fragmentthereof;

Y is selected from the group consisting of GG, SG, SS, GGG and (G)_(h)wherein h is 4, 5, 6, 7, 8, 9, or 10;

o is 0 or 1;

B is PilA from Haemophilus influenzae or an immunogenic fragmentthereof, or Protein E from Haemophilus influenzae or an immunogenicfragment thereof;

Z is GGHHHHHH (SEQ ID NO. 3); and

p is 0 or 1.

As a second aspect, the present invention provides immunogeniccompositions comprising fusion proteins of formula (I). The compositionmay further comprise a pharmaceutically acceptable adjuvant. Thecomposition may comprise an excipient.

In a third aspect, the present invention provides a method for thetreatment or prevention of a condition or disease caused wholly or inpart by Haemophilus influenzae. The method comprises administering to asubject in need thereof a therapeutically effective amount of the fusionprotein of formula (I).

In a fourth aspect, the present invention provides a method for thetreatment or prevention of otitis media. The method comprisesadministering to a subject in need thereof a therapeutically effectiveamount of the fusion protein of formula (I).

In a fifth aspect, the present invention provides a method for thetreatment or prevention of exacerbations in chronic obstructivepulmonary disease. The method comprises administering to a subject inneed thereof a therapeutically effective amount of the fusion protein offormula (I).

In a sixth aspect, the present invention provides a method for thetreatment or prevention of pneumonia. The method comprises administeringto a subject in need thereof a therapeutically effective amount of thefusion protein of formula (I).

In a seventh aspect, the present invention provides a pharmaceuticalcomposition comprising a fusion protein of formula (I) for use in thetreatment or prevention of a condition or disease caused wholly or inpart by Haemophilus influenzae. Pharmaceutical compositions may furthercomprise a pharmaceutically acceptable adjuvant.

In an eighth aspect, the present invention provides nucleic acidsencoding the proteins of the invention.

In a ninth aspect, the present invention provides a process of producingnucleic acids of the invention.

Further aspects of the present invention are described in the detaileddescription of particular embodiments, examples and claims which follow.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. SDS-PAGE of induced bacterial extracts for fusion proteinconstructs LVL291, LVL268 and LVL269. Insoluble fraction (I), Solublefraction (S) and Culture Media fraction (M) were loaded for LVL291,LVL268 and LVL269 before and after induction (ind).

FIG. 2. SDS-PAGE and Western blot related to purification extracts forfusion protein constructs LVL291, LVL268 and LVL269. Flow throughfraction (Ft), Wash fraction (W) and Elution fraction (E) were loadedfor purification of LVL291, LVL268 and LVL269. Anti-his tag was used toprobe extracts.

FIG. 3. SDS-PAGE of induced bacterial and purification extracts forfusion protein constructs LVL291 and LVL315. Culture Media fraction (M),Soluble fraction (Sol), Insoluble fraction (Ins), Flow through fraction(Ft), Wash fraction #1 (W1), Wash fraction #2 (W2) and Elution fraction(E) were loaded for LVL291 and LVL315.

FIG. 4. SDS-PAGE of induced bacterial and purification extracts forfusion protein construct LVL312. Culture Media fraction (M), Solublefraction (Sol), Insoluble fraction (Ins), Flow Through fraction (Ft),Wash fraction #1 (W1), Wash fraction #2 (W2) and Elution fraction (E)were loaded for LVL312.

FIG. 5. SDS-PAGE of induced (1 mM and 10 μM IPTG) bacterial extracts forfusion protein construct LVL317. Extracts from before (NI) and afterinduction (In), Soluble fraction (S), Insoluble fraction (I).

FIG. 6. SDS-PAGE of induced (1 mM and 10 μM IPTG) bacterial extracts forfusion protein construct LVL318. Extracts from before (NI) and afterinduction (In), Culture Media fraction (M), Soluble fraction (S),Insoluble fraction (I).

FIG. 7. CD spectra of PE, PilA and PE-PilA fusion proteins.

FIG. 8. Combination of PE and PilA CD spectrum.

FIG. 9. PilA thermal denaturation curve.

FIG. 10. PE denaturation curve.

FIG. 11. PE-PilA fusion protein thermal denaturation curve.

FIG. 12. Typical SP Sepharose™ Fast Flow chromatogram.

FIG. 13. Typical Q Sepharose™ Fast Flow chromatogram.

FIG. 14. SDS-PAGE of In-process samples from purification process ofPE-PilA fusion protein.

FIG. 15. Western Blot of In-process samples of purification process fromPE-PilA fusion protein. Blot using rabbit polyclonal anti-PE.

FIG. 16. Western Blot of In-process samples of purification process fromPE-PilA fusion protein. Blot using rabbit polyclonal anti-E. coli (BLR).

FIG. 17. Thermal transition of PE-PilA fusion protein and PE and PilAproteins. Curves: PilA (1), Protein E (Prot E, PE) (2), PE-PilA PurifiedBulk not diluted, 737 μg/ml (3), and PE-PilA Purified Bulk diluted atFinal Container concentration 60 μg/ml (4).

FIG. 18. Antibody responses against LVL291 PE-PilA fusion protein andagainst monovalent PE and PilA in the Balb/c mouse model.

FIG. 19. Effect of PE-PilA fusion protein vaccination on NTHi strain86-028NP bacterial clearance in mouse nasopharynx.

FIG. 20. Effect of PE-PilA fusion protein vaccination on NTHi strain3224A bacterial clearance in mouse nasopharynx.

FIG. 21. Effect of PilA vaccination on bacterial clearance in mousenasopharynx.

FIG. 22. Effect of PE vaccination on bacterial clearance in mousenasopharynx.

FIG. 23. (a) LVL317 PE-PilA fusion protein binding to vitronectin and(b) LVL317 and LVL735 PE-PilA fusion protein bound to vitronectin.

FIG. 24. Inhibition of vitronectin binding by polyclonal antibodiesagainst PE-PilA fusion protein.

FIG. 25. SDS-PAGE of soluble fractions of induced bacterial extracts forfusion protein constructs LVL291, LVL702, LVL736, LVL737, LVL738,LVL739, LVL740 and pET26b vector (negative control). (a) Experiment 1(b) Experiment 2 (c) Experiment 3. PE-PilA fusion protein indicated byarrow.

FIG. 26. The average band percentage of fusion protein in the solublefraction from Experiments 1, 2 and 3.

FIG. 27. PE and PilA antibody response to LVL317 and LVL735.

FIG. 28. Effect of LVL735 and LVL317 vaccination on bacterial clearancein a mouse model of non-typeable Haemophilus influenzae nasopharyngealcolonization.

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise explained or defined herein, all technical andscientific terms used herein have the same meaning as commonlyunderstood by one of ordinary skill in the art to which this disclosurebelongs. For example, definitions of common terms in molecular biologycan be found in Benjamin Lewin, Genes V, published by Oxford UniversityPress, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), TheEncyclopedia of Molecular Biology, published by Blackwell Science Ltd.,1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biologyand Biotechnology: a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8).

The singular terms “a,” “an,” and “the” include plural referents unlesscontext clearly indicates otherwise. Similarly, the word “or” isintended to include “and” unless the context clearly indicatesotherwise. It is further to be understood that all base sizes or aminoacid sizes, and all molecular weight or molecular mass values, given fornucleic acids or polypeptides are approximate, and are provided fordescription. Additionally, numerical limitations given with respect toconcentrations or levels of a substance, such as an antigen may beapproximate. Thus, where a concentration is indicated to be (forexample) approximately 200 pg, it is intended that the concentrationincludes values slightly more or slightly less than (“about” or “˜”) 200pg.

Although methods and materials similar or equivalent to those describedherein can be used in the practice or testing of this disclosure,suitable methods and materials are described below.

The term “comprises” means “includes”. Thus, unless the context requiresotherwise, the word “comprises,” and variations such as “comprise” and“comprising” will be understood to imply the inclusion of a statedcompound or composition (e.g., nucleic acid, polypeptide, antigen) orstep, or group of compounds or steps, but not to the exclusion of anyother compounds, composition, steps, or groups thereof. Theabbreviation, “e.g.” is derived from the Latin exempli gratia, and isused herein to indicate a non-limiting example. Thus, the abbreviation“e.g.” is synonymous with the term “for example.”

In order to facilitate review of the various embodiments of thisdisclosure, the following explanations of terms are provided. Additionalterms and explanations are provided in the context of this disclosure.

A “subject” as used herein is a mammal, including humans, non-humanprimates, and non-primate mammals such as members of the rodent genus(including but not limited to mice and rats) and members of the orderLagomorpha (including but not limited to rabbits).

As used herein “Protein E”, “protein E”, “Prot E”, and “PE” mean ProteinE from H. influenzae. Protein E may consist of or comprise the aminoacid sequence of SEQ ID NO. 4 (MKKIILTLSL GLLTACSAQI QKAEQNDVKLAPPTDVRSGY IRLVKNVNYY IDSESIWVDN QEPQIVHFDA VVNLDKGLYV YPEPKRYARSVRQYKILNCA NYHLTQVRTD FYDEFWGQGL RAAPKKQKKH TLSLTPDTTL YNAAQIICANYGEAFSVDKK) as well as sequences with at least or exactly 75%, 77%, 80%,85%, 90%, 95%, 97%, 99% or 100% identity, over the entire length, to SEQID NO. 4. Comparison of 53 sequences of Protein E from Haemophilusinfluenzae (Table 1, SEQ ID NO. 5-SEQ ID NO. 57) demonstratedapproximately 77% to approximately 100% identity to Protein E as setforth in SEQ ID NO. 4. For example, in the amino acid sequence ofProtein E, amino acid #20 may be isoleucine (I) or threonine (T); aminoacid #23 may be alanine (A) or valine (V); amino acid #24 may be lysine(K) or glutamic acid (E); amino acid #31 may be alanine (A) or threonine(T); amino acid #32 may be proline (P) or alanine (A); amino acid #34may be threonine (T) or alanine (A); amino acid #37 may be arginine (R)or glutamine (Q); amino acid #47 may be valine (V) or alanine (A); aminoacid #57 may be tryptophane (W) or may be absent (−); amino acid #70 maybe alanine (A) or threonine (T); amino acid #93 may be glutamine (Q) orabsent (−); amino acid #109 may be threonine (T) or isoleucine (I);amino acid #119 may be glycine (G) or serine (S); amino acid #153 may beglutamic acid (E) or lysine (K); amino acid #156 may be serine (S) orleucine (L); amino acid #160 may be lysine (K) or asparagine (N); aminoacid #161 may be lysine (K), isoleucine (I) or absent (−); amino acids#162-#195 may be absent, or as set forth in SEQ ID NO. 15 (with (−)indicating amino acid #166 is absent) or as set forth in SEQ ID NO. 16;or any combination thereof.

Protein E may consist of or comprise an amino acid sequence that differsfrom SEQ ID NO. 4 at any one or more amino acid selected from the groupconsisting of: amino acid #20, amino acid #23, amino acid #24, aminoacid #31, amino acid #32, amino acid #34, amino acid #37, amino acid#47, amino acid #57, amino acid #70, amino acid #93, amino acid #109,amino acid #119, amino acid #153, amino acid #156, amino acid #160,amino acid #161 and amino acids #162-#195, wherein amino acid #20 isthreonine (T); amino acid #23 is valine (V); amino acid #24 is lysine(K); amino acid #31 is threonine (T); amino acid #32 is alanine (A);amino acid #34 is alanine (A); amino acid #37 is glutamine (Q); aminoacid #47 is alanine (A); amino acid #57 is absent (−); amino acid #70 isthreonine (T); amino acid #93 is absent (−); amino acid #109 isisoleucine (I); amino acid #119 is serine (S); amino acid #153 is lysine(K); amino acid #156 is leucine (L); amino acid #160 is asparagine (N);amino acid #161 is lysine (K) or isoleucine (I); or amino acids#162-#195 are as set forth in SEQ ID NO. 15 (with (−) indicating aminoacid #166 is absent) or as set forth in SEQ ID NO. 16.

TABLE 1Protein E amino add sequences from 53 strains of Haemophilus influenzae(SEQ ID NO. 5-SEQ ID NO. 57). -indicates amino acid is absent.Strain Name Protein E sequence 3224AMKKIILTLSLGLLTACSAQIQKAKQNDVKLAPPTDVRSGYIRLVKNVNYYIDSESIWVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPKKQKKHTLSLTPDTTLYNAAQIICANYGEAFSVDKK (SEQ ID NO. 5) RdKW20MKKIILTLSLGLLTACSAQIQKAEQNDVKLAPPTDVRSGYIRLVKNVNYYIDSESIWVDNQEPQIVHFDAVVNLDRGLYVYPEPKRYARSVRQYKILNCANYHLTQIRTDFYDEFWGQGLRAAPKKQKKHTLSLTPDTTLYNAAQIICANYGKAFSVDKK (SEQ ID NO. 6) 86-028NPMKKIILTLSLGLLTACSAQIQKAKQNDVKLAPPTDVRSGYIRLVKNVNYYIDSESIWVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPKKQKKHTLSLTPDTTLYNAAQIICANYGEAFSVDKK (SEQ ID NO. 7) R2846MKKIILTLSLGLLTACSAQIQKAEQNDVKLAPPTDVRSGYIRLVKNVNYYIDSESIWVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPKKQKKHTLSLTPDTTLYNAAQIICANYGKAFSVDKK (SEQ ID NO. 8) R2866MKKIILTLSLGLLTACSAQIQKAEQNDVKLAPPTDVRSGYIRLVKNVNYYIDSESIWVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPKKQKKHTLSLTPDTTLYNAAQIICANYGKAFSVDKK (SEQ ID NO. 9) 3655MKKIILTLSLGLLTACSAQIQKAEQNDMKLAPPTDVRSGYIRLVKNVNYYIDSESIWVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPKKQKKHTLSLTPDTTLYNAAQIICANYGKAFSVDKK (SEQ ID NO. 10) PittAAMKKIILTLSLGLLTACSAQIQKAKQNDVKLAPPTDVRSGYIRLVKNVNYYIDSESIWVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPKKQKKHTLSLTPDTTLYNAAQIICANYGEAFSVDKK (SEQ ID NO. 11) PittEEMKKIILTLSLGLLTACSAQIQKAEQNDMKLAPPTDVRSGYIRLVKNVNYYIDSESI-VDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPKKQKKHTLSLTPDTTLYNAAQIICANYGKAFSVDKK(SEQ ID NO. 12) PittHHMKKIILTLSLGLLTACSAQIQKAEQNDVKLAPPTDVRSGYIRLVKNVNYYIDSESIWVDNQEPQIVHFDTVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPKKQKKHTLSLTPDTTLYNAAQIICANYGKAFSVDKK (SEQ ID NO. 13) PittIIMKKIILTLSLGLLTACSAQIQKAEQNDVKLAPPTDVRSGYIRLVKNVNYYIDSESIWVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPKKQKKHTLSLTPDTTLYNAAQIICANYGKAFSVDKK (SEQ ID NO. 14) R3021MKKIILTLSLGLLTACSAQTQKAEQNDVKLTPPTDVQSGYVRLVKNVNYYIDSESIWVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRIDEYDEFWGQGLRAAPKKQKKHTLSLTPDTTLYNAAQIICANYGKAFSVDKNKKICT-LISLNFIQLLGCREYSIFLQLLLFYCWHF (SEQ ID NO. 15) 22.4-21MKKIILTLSLGLLTACSAQIQKAEQNDVKLAPPTDVRSGYIRLVKNVNYYIDSESIWVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPKKQKKHTLSLTPDTTLYNAAQIICANYGKAFSVDKKIKKICTLISLNFIQLLGCREYSIFLQLLLFYCWHF (SEQ ID NO. 16) 3219CMKKIILTLSLGLLTACSAQIQKAEQNDMKLAPPTDVRSGYIRLVKNVNYYIDSESIWVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPKKQKKHTLSLTPDTTLYNAAQIICANYGKAFSVDKK (SEQ ID NO. 17) 3185MKKIILTLSLGLLTACSAQIQKAEQNDVKLAPPTDVRSGYIRLVKNVNYYIDSESIWVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPKKQKKHTLSLTPDTTLYNAAQIICANYGKAFSVDKK (SEQ ID NO. 18) 3241AMKKIILTLSLGLLTACSAQIQKAKQNDVKLAPPTDVRSGYIRLVKNVNYYIDSESIWVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPKKQKKHTLSLTPDTTLYNAAQIICANYGEAFSVDKK (SEQ ID NO. 19) 038144S1MKKIILTLSLGLLTACSAQTQKVEQNDVKLTAPTDVRSGFVRLVKNVNYYIDSESIWVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPKKQKKHTLSLTPDTTLYNAAQIICANYGKAFLVDKK (SEQ ID NO. 20) 810956MKKIILTLSLGLLTACSAQIQKAKQNDVKLAPPTDVRSGYIRLVKNVNYYIDSESIWVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPKKQKKHTLSLTPDTTLYNAAQIICANYGEAFSVDKK (SEQ ID NO. 21) 821246MKKIILTLSLGLLTACSAQIQKAEQNDVKLAPPTDVRSGYIRLVKNVNYYIDSESIWVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQIRTDFYDEFWGQGLRAAPKKQKKHTLSLTPDTTLYNAAQIICANYGKAFSVDKK (SEQ ID NO. 22) 840645MKKIILTLSLGLLTACSAQIQKAKQNDVKLAPPTDVRSGYIRLVKNVNYYIDSESIWVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPKKQKKHTLSLTPDTTLYNAAQIICANYGEAFSVDKK (SEQ ID NO. 23) 902550Z19MKKIILTLSLGLLTACSAQTQKVEQNDVKLTPPTDVRSGYVRLVKNVNYYIDSESIWVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPKKQKKHTLSLTPDTTLYNAAQIICANYGKAFSVDKK (SEQ ID NO. 24) A840177MKKIILTLSLGLLTACSAQIQKAKQNDVKLAPPTDVRSGYIRLVKNVNYYIDSESIWVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPKKQKKHTLSLTPDTTLYNAAQIICANYGEAFSVDKK (SEQ ID NO. 25) A860514MKKIILTLSLGLLTACSAQTQKVEQNDVKLTAPTDVRSGYVRLVKNANYYIDSESIWVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPKKQKKHTLSLTPDTTLYNAAQIICANYGKAFSVDKK (SEQ ID NO. 26) A950014MKKIILTLSLGLLTACSAQIQKAEQNDVKLAPPTDVRSGYIRLVKNVNYYIDSESIWVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRIDEYDEFWGQGLRAAPKKQKKHTLSLTPDTTLYNAAQIICANYGKAFSVDKK (SEQ ID NO. 27) 306543X4MKKIILTLSLGLLTACSAQIQKAEQNDVKLAPPTDVRSGYIRLVKNVNYYIDSESIWVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPKKQKKHTLSLTPDTTLYNAAQIICANYGKAFSVDKK(SEQ ID NO. 28) A930105MKKIILTLSLGLLTACSAQIQKAEQNDVKLAPPTDVRSGYIRLVKNVNYYIDSESIWVDNQEPQIVHFDTVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPKKQKKHTLSLTPDTTLYNAAQIICANYGKAFSVDKK(SEQ ID NO. 29) 901905UMKKIILTLSLGLLTACSAQIQKAEQNDVKLAPPTDVRSGYIRLVKNVNYYIDSESIWVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPKKQKKHTLSLTPDTTLYNAAQIICANYGKAFSVDKK (SEQ ID NO. 30) A920030MKKIILTLSLGLLTACSAQIQKAKQNDVKLAPPTDVRSGYIRLVKNVNYYIDSESIWVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPKKQKKHTLSLTPDTTLYNAAQIICANYGEAFSVDKK (SEQ ID NO. 31) 3221BMKKIILTLSLGLLTACSAQIQKAEQNDVKLAPPTDVRSGYIRLVKNVNYYIDSESIWVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPKKQKKHTLSLTPDTTLYNAAQIICANYGKAFSVDKK (SEQ ID NO. 32) 27W116791NMKKIILTLSLGLLTACSAQTQKVEQNDVKLTPPTDVRSGYVRLVKNVNYYIDSESIWVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPKKQKKHTLSLTPDTTLYNAAQIICANYGKAFSVDKK (SEQ ID NO. 33) N218MKKIILTLSLGLLTACSAQIQKAEQNDVKLAPPTDVRSGYIRLVKNVNYYIDSESIWVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPKKQKKHTLSLTPDTTLYNAAQIICANYGKAFSVDKK (SEQ ID NO. 34) N163MKKIILTLSLGLLTACSAQIQKAKQNDVKLAPPTDVRSGYIRLVKNVNYYIDSESIWVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPKKQKKHTLSLTPDTTLYNAAQIICANYGEAFSVDKK (SEQ ID NO. 35) N162MKKIILTLSLGLLTACSAQIQKAKQNDVKLAPPTDVRSGYIRLVKNVNYYIDSESIWVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPKKQKKHTLSLTPDTTLYNAAQIICANYGEAFSVDKK (SEQ ID NO. 36) N107MKKIILTLSLGLLTACSAQTQKAEQNDVKLAPPTDVRSGYIRLVKNVNYYIDSESIWVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQIRTDFYDEFWGQGLRAAPKKQKKHTLSLTPDTTLYNAAQIICANYGKAFSVDKK (SEQ ID NO. 37) N91MKKIILTLSLGLLTACSAQTQKVEQNDVKLTAPADVRSGYVRLVKNVNYYIDSESIWVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPKKQKKHTLSLTPDTTLYNAAQIICANYGKAFSVDKK (SEQ ID NO. 38) D211PGMKKIILTLSLGLLTACSAQIQKAKQNDVKLAPPTDVRSGYIRLVKNVNYYIDSESIWVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVR-YKILNCANYHLTQVRTDFYDEFWGQGLRAAPKKQKKHTLSLTPDTTLYNAAQIICANYGEAFSVDKK (SEQ ID NO. 39) D211PDMKKIILTLSLGLLTACSAQIQKAKQNDVKLAPPTDVRSGYIRLVKNVNYYIDSESIWVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVR-YKILNCANYHLTQVRTDFYDEFWGQGLRAAPKKQKKHTLSLTPDTTLYNAAQIICANYGEAFSVDKK (SEQ ID NO. 40) D201PGMKKIILTLSLGLLTACSAQIQKAEQNDVKLAPPTDVRSGYIRLVKNVNYYIDSESIWVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPKKQKKHTLSLTPDTTLYNAAQIICANYGKAFSVDKK (SEQ ID NO. 41) D201PDMKKIILTLSLGLLTACSAQIQKAEQNDVKLAPPTDVRSGYIRLVKNVNYYIDSESIWVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPKKQKKHTLSLTPDTTLYNAAQIICANYGKAFSVDKK (SEQ ID NO. 42) D198PGMKKIILTLSLGLLTACSAQIQKAEQNDVKLAPPTDVRSGYIRLVKNVNYYIDSESIWVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPKKQKKHTLSLTPDTTLYNAAQIICANYGKAFSVDKK (SEQ ID NO. 43) D198PDMKKIILTLSLGLLTACSAQIQKAEQNDVKLAPPTDVRSGYIRLVKNVNYYIDSESIWVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPKKQKKHTLSLTPDTTLYNAAQIICANYGKAFSVDKK (SEQ ID NO. 44) D195PDMKKIILTLSLGLLTACSAQIQKAEQNDVKLAPPTDVRSGYIRLVKNVNYYIDSESIWVDNQEPQIVHFDTVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRTDFYDEFWGQSLRAAPKKQKKHTLSLTPDTTLYNAAQIICANYGKAFSVDKK (SEQ ID NO. 45) D189PGMKKIILTLSLGLLTACSAQTQKVEQNDVKLTPPTDVRSGYVRLVKNVNYYIDSESIWVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPKKQKKHTLSLTPDTTVYNAAQIICANYGKAFSVDKK (SEQ ID NO. 46) D189PDMKKIILTLSLGLLTACSAQTQKVEQNDVKLTPPTDVRSGYVRLVKNVNYYIDSESIWVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPKKQKKHTLSLTPDTTVYNAAQIICANYGKAFSVDKK (SEQ ID NO. 47) D129CGMKKIILTLSLGLLTACSAQIQKAKQNDVKLAPPTDVRSGYIRLVKNVNYYIDSESIWVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPKKQKKHTLSLTPDTTLYNAAQIICANYGEAFSVDKK (SEQ ID NO. 48) D124PGMKKIILTLSLGLLTACSAQIQKAEQNDVKLAPPTDVRSGYIRLVKNVNYYIDSESIWVDNQEPQIVHFDTVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPKKQKKHTLSLTPDTTLYNAAQIICANYGKAFSVDKK (SEQ ID NO. 49) D124PDMKKIILTLSLGLLTACSAQIQKAEQNDVKLAPPTDVRSGYIRLVKNVNYYIDSESIWVDNQEPQIVHFDTVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPKKQKKHTLSLTPDTTLYNAAQIICANYGKAFSVDKK(SEQ ID NO. 50) D58PGMKKIILTLSLGLLTACSAQTQKAEQNDVKLTPPTDVRSGYIRLVKNVNYYIDSESIWVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPKKQKKHTLSLTPDTTLYNAAQIICANYGKAFSVDKK (SEQ ID NO. 51) D330DMKKIILTLSLGLLTACSAQIQKAKQNDVKLAPPTDVRSGYIRLVKNVNYYIDSESIWVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPKKQKKHTLSLTPDTTLYNAAQIICANYGKAFSVDKK (SEQ ID NO. 52) BS433MKKIILTLSLGLLTACSAQIQKAEQNDVKLAPPTDVRSGYIRLVKNVNYYIDSESIWVDNQEPQIVHFDTVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPKKQKKHTLSLTPDTTLYNAAQIICANYGKAFSVDKK (SEQ ID NO. 53) BS432MKKIILTLSLGLLTACSAQTQKAEQNDVKLAPPTDVRSGYIRLVKNVNYYIDSESIWVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQIRTDFYDEFWGQGLRAAPKKQKKHTLSLTPDTTLYNAAQIICANYGKAFSVDKK(SEQ ID NO. 54) 1714MKKIILTLSLGLLTACSAQIQKAKQNDVKLAPPTDVRSGYIRLVKNVNYYIDSESIWVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPKKQKKHTLSLTPDTTLYNAAQIICANYGEAFSVDKK (SEQ ID NO. 55) 1128MKKIILTLSLGLLTACSAQIQKAEQNDVKLAPPTDVRSGYIRLVKNVNYYIDSESIWVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPKKQKKHTLSLTPDTTLYNAAQIICANYGKAFSVDKK (SEQ ID NO. 56) BS430MKKIILTLSLGLLTACSAQIQKAEQNDMKLAPPTDVRSGYIRLVKNVNYYIDSESI-VDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPKKQKKHTLSLTPDTTLYNAAQIICANYGKAFSVDKK (SEQ ID NO. 57)

Protein E may be Protein E from H. influenzae strain 3224A, RdKW20,86-028NP, R2846, R2866, 3655, PittAA, PittEE, PittHH, PittII, R3021,22.4-21, 3219C, 3185, 3241A, 038144S1, 810956, 821246, 840645,902550Z19, A840177, A860514, A950014, 306543X4, A930105, 901905U,A920030, 3221B, 27W116791N, N218, N163, N162, N107, N91, D211PG, D211PD,D201PG, D201PD, D198PG, D198PD, D195PD, D189PG, D189PD, D129CG, D124PG,D124PD, D58PG, D330D, BS433, BS432, 1714, 1128 or BS430. Protein E maybe Protein E as set forth in any of SEQ ID NO. 5-SEQ ID NO. 57.

Protein E may be a sequence with at least 95% identity, over the entirelength, to any of SEQ ID NO. 4-SEQ ID NO. 57. Protein E may be asequence with at least 95% identity, over the entire length, to any ofthe sequences set forth in Table 1, SEQ ID NO. 5-SEQ ID NO. 57.

Immunogenic fragments of Protein E comprise immunogenic fragments of atleast 7, 10, 15, 20, 25, 30 or 50 contiguous amino acids of SEQ ID NO.4. The immunogenic fragments may elicit antibodies which can bind SEQ IDNO. 4.

Immunogenic fragments of Protein E may comprise immunogenic fragments ofat least 7, 10, 15, 20, 25, 30 or 50 contiguous amino acids of any ofSEQ ID NO. 4-SEQ ID NO. 57. The immunogenic fragments may elicitantibodies which can bind the full length sequence from which thefragment is derived.

Immunogenic fragments of Protein E comprise immunogenic fragments of atleast 7, 10, 15, 20, 25, 30 or 50 contiguous amino acids of SEQ ID NO.5-SEQ ID NO. 57. The immunogenic fragments may elicit antibodies whichcan bind the full length sequence from which the fragment is derived.

As used herein “PilA” means Pilin A from H. influenzae. PilA may consistof or comprise the protein sequence of SEQ ID NO. 58 (MKLTTQQTLKKGFTLIELMI VIAIIAILAT IAIPSYQNYT KKAAVSELLQ ASAPYKADVE LCVYSTNETTNCTGGKNGIA ADITTAKGYV KSVTTSNGAI TVKGDGTLAN MEYILQATGN AATGVTWTTTCKGTDASLFP ANFCGSVTQ) as well as sequences with 80% to 100% identity toSEQ ID NO. 58. For example, PilA may be at least 80%, 85%, 90%, 95%, 97%or 100% identical to SEQ ID NO. 58. Full length comparison of 64sequences of PilA from Haemophilus influenzae (Table 2, SEQ ID NO.58-SEQ ID NO. 121) demonstrated approximately 80% to 100% identity toPilA as set forth in SEQ ID NO. 58. For example, in the amino acidsequence of PilA, amino acid #6 may be glutamine (Q) or leucine (L);amino acid #7 may be glutamine (Q) or threonine (T); amino acid #37 maybe glutamine (Q) or lysine (K); amino acid #44 may be alanine (A) orserine (S); amino acid #57 may be alanine (A) or serine (S); amino acid#67 may be asparagine (N) or glycine (G); amino acid #68 may be glutamicacid (E) or lysine (K); amino acid #69 may be theronine (T) or proline(P); amino acid #71 may be lysine (K), asparagine (N), serine (S) orthreonine (T); amino acid #73 may be threonine (T), serine (S) ormethionine (M); amino acid #76 may be lysine (K), serine (S) orasparagine (N); amino acid #84 may be threonine (T) or lysine (K); aminoacid #86 may be alanine (A) or valine (V); amino acid #91 may be lysine(K) or alanine (A); amino acid #94 may be threonine (T), isoleucine (I)or lysine (K); amino acid #96 may be serine (S) or glutamine (Q); aminoacid #97 may be asparagine (N) or serine (S); amino acid #99 may bealanine (A) or glycine (G); amino acid #103 may be alanine (A) or lysine(K); amino acid #109 may be aspartic acid (D), alanine (A) or threonine(T); amino acid #110 may be glycine (G), asparagine (N), or arginine(R); amino acid #112 may be serine (S) or glutamic acid (E); amino acid#114 may be threonine (T) or isoleucine (I); amino acid #116 may bethreonine (T) or glutamine (Q); amino acid #118 may be glutamic acid(E), threonine (T), alanine (A), lysine (K) or serine (S); amino acid#121 may be serine (S) or alanine (A); amino acid #122 may be alanine(A) or threonine (T); amino acid #123 may be lysine (K), threonine (T)or alanine (A); amino acid #128 may be lysine (K) or threonine (T);amino acid #135 may be aspartic acid (D) or glutamic acid (E); aminoacid #136 may be alanine (A) or threonine (T); amino acid #145 may beglycine (G) or arginine (R); amino acid #149 may be glutamine (Q) orlysine (K); or any combination thereof.

Pil A may consist of or comprise an amino acid sequence that differsfrom SEQ ID NO. 58 at any or more amino acid selected from the groupconsisting of amino acid #6, amino acid #7, amino acid #37, amino acid#44, amino acid #57, amino acid #67, amino acid #68, amino acid #69,amino acid #71, amino acid #73, amino acid #76, amino acid #84, aminoacid #86, amino acid #91, amino acid #94, amino acid #96, amino acid#97, amino acid #99, amino acid #103, amino acid #109, amino acid #110,amino acid #112, amino acid #114, amino acid #116, amino acid #118 aminoacid, #121, amino acid #122, amino acid #123, amino acid #128, aminoacid #135, amino acid #136, amino acid #145 and amino acid #149, whereinamino acid #6 is leucine (L); amino acid #7 is threonine (T); amino acid#37 is lysine (K); amino acid #44 is serine (S); amino acid #57 isserine (S); amino acid #67 is glycine (G); amino acid #68 is lysine (K);amino acid #69 is proline (P); amino acid #71 is lysine (K), serine (S)or threonine (T); amino acid #73 is serine (S) or methionine (M); aminoacid #76 is serine (S) or asparagine (N); amino acid #84 is lysine (K);amino acid #86 is valine (V); amino acid #91 is alanine (A); amino acid#94 is isoleucine (I) or lysine (K); amino acid #96 is glutamine (Q);amino acid #97 is serine (S); amino acid #99 is glycine (G); amino acid#103 is alanine (A); amino acid #109 is aspartic acid (D) or threonine(T); amino acid #110 is glycine (G) or arginine (R); amino acid #112 isserine (S); amino acid #114 is threonine (T); amino acid #116 isthreonine (T); amino acid #118 is glutamic acid (E), alanine (A), lysine(K) or serine (S); amino acid #121 is serine (S); amino acid #122 isthreonine (T); amino acid #123 is lysine (K) or alanine (A); amino acid#128 is lysine (K); amino acid #135 is glutamic acid (E); amino acid#136 is threonine (T); amino acid #145 is arginine (R); amino acid #149is lysine (K).

TABLE 2Pilin A amino acid sequences from 64 strains of Haemophilus influenzae (SEQID NO. 58-SEQ ID NO. 121). Strain Name PilA sequence 86-028NPMKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTNETTNCTGGKNGIAADITTAKGYVKSVTTSNGAITVKGDGTLANMEYILQATGNAATGVTWTTTCKGTDASLFPANFCGSVTQ (SEQ ID NO. 58) NTHi3219CMKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTNETTKCTGGKNGIAADITTAKGYVKSVTTSNGAITVAGNGTLDGMSYTLTAEGDSAKGVTWKTTCKGTDASLFPANFCGSVTQ (SEQ ID NO. 59) NTHi3224AMKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTNETTNCTGGKNGIAADITTAKGYVKSVTTSNGAITVKGDGTLANMEYILQATGNAATGVTWTTTCKGTDASLFPANFCGSVTQ (SEQ ID NO. 60) NTHi12MKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYKNYTKKAAVSELLQASAPYKADVELCVYSTGKPSSCSGGSNGIAADITTAKGYVASVITQSGGITVKGDGTLANMEYILQAAGNAAAGVTWTTTCKGTDASLFPANFCGSVTQ (SEQ ID NO. 61) NTHi44MKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTNETTNCTGGKNGIAADITTAKGYVKSVTTSNGAITVKGDGTLANMEYILQATGNAATGVTWTTTCKGTDASLFPANFCGSVTQ (SEQ ID NO. 62) NTHi67MKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKSDVELCVYSTGKPSTCSGGSNGIAADITTVKGYVKSVTTSNGAITVAGNGTLDGMSYTLTAEGDSAKGVTWTTTCKGTDASLFPANFCGSVTQ (SEQ ID NO. 63) 1054MEEMKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTNETTNCTGGKNGIAADITTAKGYVKSVTTSNGAITVKGDGTLANMEYILQATGNAATGVTWTTTCKGTDASLFPANFCGSVTQ (SEQ ID NO. 64) 1729MEEMKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTNETTNCTGGKNGIAADITTAKGYVKSVTTSNGAITVKGDGTLANMEYILQATGNAATGVTWTTTCKGTDASLFPANFCGSVTQ (SEQ ID NO. 65) 1728MEEMKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTNETTNCTGGKNGIAADITTAKGYVKSVTTSNGAITVKGDGTLANMEYILQATGNAATGVTWTTTCKGTDASLFPANFCGSVTQ (SEQ ID NO. 66) 1885MEEMKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYKNYTKKAAVSELLQASAPYKADVELCVYSTNEITNCMGGKNGIAADITTAKGYVASVKTQSGGITVKGDGTLANMEYILQATGNAAAGVTWTTTCKGTDASLFPANFCGSITQ (SEQ ID NO. 67) 1060MEEMKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKASVSELLQASAPYKADVELCVYSTNETTNCTGGKNGIAADITTAKGYVASVKTQSGGITVKGNGTLANMEYILQAKGNATAGVTWTTTCKGTDASLFPANFCRSVTK (SEQ ID NO. 68) RdKW20MKLTTLQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTNETTSCTGGKNGIAADIKTAKGYVASVITQSGGITVKGNGTLANMEYILQAKGNAAAGVTWTTTCKGTDASLFPANFCGSVTK (SEQ ID NO. 69) 214NPMKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTGKPSSCSGGSNGIAADITTAKGYVKSVTTSNGAITVKGDGTLANMEYILQASGNAATGVTWTTTCKGTDASLFPANFCGSVTQ (SEQ ID NO. 70) 1236MEEMKLTTLQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTNETTSCTGGKNGIAADIKTAKGYVASVITQSGGITVKGNGTLANMEYILQAKGNAAAGVTWTTTCKGTDASLFPANFCGSVTK (SEQ ID NO. 71) 1714MEEMKLTTLQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTGKPSTCSGGSNGIAADITTAKGYVASVKTQSGGITVKGNGTLANMEYILQATGNAATGVTWTTTCKGTDASLFPANFCGSVTQ (SEQ ID NO. 72) 1128MEEMKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKASVSELLQASAPYKSDVELCVYSTGKPSTCSGGSNGIAADITTAKGYVASVKTQSGGITVKGNGTLANMEYILQAKGNATAGVTWTTTCKGTDASLFPANFCRSVTK (SEQ ID NO. 73) R2846MKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTNETTNCTGGKNGIAADITTAKGYVKSVTTSNGAITVKGDGTLANMEYILQATGNAATGVTWTTTCKGTDASLFPANFCGSVTQ (SEQ ID NO. 74) R2866MKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTNETTNCTGGKNGIAADITTAKGYVASVKTQSGGITVKGDGTLANMEYILQATGNAATGVTWTTTCKGTEASLFPANFCGSVTQ (SEQ ID NO. 75) 3655MKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKASVSELLQASAPYKADVELCVYSTNETTNCTGGKNGIAADITTAKGYVASVKTQSGGITVKGNGTLANMEYILQAKGNATAGVTWTTTCKGTDASLFPANFCRSVTK (SEQ ID NO. 76) PittAAMKLTTLQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTGKPSTCSGGSNGIAADITTAKGYVASVKTQSGGITVKGNGTLANMEYILQATGNAATGVTWTTTCKGTDASLFPANFCGSVTQ (SEQ ID NO. 77) PittGGMKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTGKPSTCSGGSNGIAADITTAKGYVASVKTQSGGITVKGNGTLANMEYILQAKGNATAGVTWTTTCKGTDASLFPANFCRSVTK (SEQ ID NO. 78) PittIIMKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTNETTNCTGGKNGIAADITTAKGYVASVKTQSGGITVKGDGTLANMEYILQATGNAATGVTWTTTCKGTEASLFPANFCGSVTQ (SEQ ID NO. 79) R3021MKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTNETTNCTGGKNGIAADITTAKGYVASVKTQSGGITVKGDGTLANMEYILQATGNAATGVTWTTTCKGTEASLFPANFCGSVTQ (SEQ ID NO. 80) 22.4-21MKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKSDVELCVYSTGKPSTCSGGSNGIAADITTAKGYVKSVTTSNGAITVAGNGTLDGMSYTLTAEGDSAKGVTWKTTCKGTDASLFPANFCGSVTK (SEQ ID NO. 81) 3185AMKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTNEATKCTGGKNGIAADITTAKGYVKSVTTSNGAITVKGDGTLANMEYILQASGNAATGVTWTTTCKGTDASLFPANFCGSVTQ (SEQ ID NO. 82) 3221BMKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTNEATKCTGGKNGIAADITTAKGYVKSVTTSNGAITVKGDGTLANMEYILQASGNAATGVTWTTTCKGTDASLFPANFCGSVTQ (SEQ ID NO. 83) 3241AMKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTNETTNCTGGKNGIAADITTAKGYVKSVTTSNGAITVKGDGTLANMEYILQATGNAATGVTWTTTCKGTDASLFPANFCGSVTQ (SEQ ID NO. 84) 038144S1MKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAISELLQASAPYKSDVELCVYSTGKPSTCSGGSNGIAADITTAKGYVASVKTQSGGITVKGNGTLANMEYILQAKGNATAGVTWTTTCKGTDASLFPANFCRSVTK (SEQ ID NO. 85) 821246MKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTNETTNCTGGKNGIAADITTAKGYVASVKTQSGGITVKGDGTLANMEYILQATGNAATGVTWTTTCKGTEASLFPANFCGSVTQ (SEQ ID NO. 86) 840645MKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTNETTNCTGGKNGIAADITTAKGYVKSVTTSNGAITVKGDGTLANMEYILQATGNAATGVTWTTTCKGTDASLFPANFCGSVTQ (SEQ ID NO. 87) 902550Z19MKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKSDVELCVYSTGKPSTCSGGSNGIAADITTVKGYVKSVTTSNGAITVAGNGTLDGMSYTLTAEGDSAKGVTWTTTCKGTDASLFPANFCGSVTQ (SEQ ID NO. 88) A840177MKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTNETTNCTGGKNGIAADITTAKGYVKSVTTSNGAITVKGDGTLANMEYILQATGNAATGVTWTTTCKGTDASLFPANFCGSVTQ (SEQ ID NO. 89) A920030MKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTNETTNCTGGKNGIAADITTAKGYVASVKTQSGGITVKGNGTLANMEYILQATGNAATGVTWTTTCKGTDASLFPANFCGSVTQ (SEQ ID NO. 90) A950014MKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTGKPSTCSGGSNGIAADITTAKGYVKSVTTSNGAITVAGNGTLDRMSYTLTAEGDSAKGVTWTTTCKGTDASLFPANFCGSVTQ (SEQ ID NO. 91) 901905UMKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTGKPSSCSGGSNGIAADITTAKGYVKSVTTSNGAITVKGDGTLANMEYILQASGNAATGVTWTTTCKGTDASLFPANFCGSVTQ (SEQ ID NO. 92) A920029MKLTTQTTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKSDVELCVYSTNETTNCTGGKNGIAADITTAKGYVASVITQSGGITVKGNGTLTNMEYILQATGNAATGVTWTTTCKGTDASLFPANFCGSITQ (SEQ ID NO. 93) A930105MKLTTLQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTGKPSTCSGGNNGIAADIKTAKGYVASVKTQSGGITVKGDGTLANMEYILQATGNAATGVTWTTTCKGTDASLFPANFCGSVTQ (SEQ ID NO. 94) 306543X4MKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTGKPSSCSGGSNGIAADITTAKGYVKSVTTSNGAITVKGDGTLANMEYILQASGNAATGVTWTTTCKGTDASLFPANFCGSVTQ (SEQ ID NO. 95) N218MKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTNEATKCTGGKNGIAADITTAKGYVKSVTTSNGAITVKGDGTLANMEYILQASGNAATGVTWTTTCKGTDTSLFPANFCGSVTQ (SEQ ID NO. 96) N163MKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTNETTNCTGGKNGIAADITTAKGYVKSVTTSNGAITVKGDGTLANMEYILQATGNAATGVTWTTTCKGTDASLFPANFCGSVTQ (SEQ ID NO. 97) N162MKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTNETTNCTGGKNGIAADITTAKGYVASVKTQSGGITVKGNGTLANMEYILQATGNAATGVTWTTTCKGTDASLFPANFCGSVTQ (SEQ ID NO. 98) N120MKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTGKPSTCSGGSNGIAADITTAKGYVASVKTQSGGITVKGNGTLANMEYILQAKGNATAGVTWTTTCKGTDASLFPANFCRSVTK (SEQ ID NO. 99) N107MKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTGKPSTCSGGSNGIAADITTAKGYVASVKTQSGGITVKGNGTLANMEYILQAKGNATAGVTWTTTCKGTDASLFPANFCRSVTK (SEQ ID NO. 100) N92MKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTNETTNCTGGKNGIAADITTAKGYVKSVTTSNGAITVKGDGTLANMEYILQATGNAATGVTWTTTCKGTDASLFPANFCGSVTQ (SEQ ID NO. 101) N91MKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTGKPSTCSGGSNGIAADITTAKGYVASVKTQSGGITVKGNGTLANMEYILQAKGNATAGVTWTTTCKGTDASLFPANFCRSVTK (SEQ ID NO. 102) D219PGMKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTNEATKCTGGKNGIAADITTAKGYVKSVTTSNGAITVKGDGTLANMEYILQASGNAATGVTWTTTCKGTDASLFPANFCGSVTQ (SEQ ID NO. 103) D211PGMKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTNETTNCTGGKNGIAADITTAKGYVKSVTTSNGAITVKGDGTLANMEYILQATGNAATGVTWTTTCKGTDASLFPANFCGSVTQ (SEQ ID NO. 104) D211PDMKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTNETTNCTGGKNGIAADITTAKGYVKSVTTSNGAITVKGDGTLANMEYILQATGNAATGVTWTTTCKGTDASLFPANFCGSVTQ (SEQ ID NO. 105) D204CDMKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTNETTNCTGGKNGIAADITTAKGYVKSVTTSNGAITVKGDGTLANMEYILXATGNAATGVTWTTTCKGTDASLFPANFCGSVTQ (SEQ ID NO. 106) D198PGMKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTNETTNCTGGKNGIAADITTAKGYVKSVTTSNGAITVKGDGTLANMEYILQATGNAATGVTWTTTCKGTDASLFPANFCGSVTQ (SEQ ID NO. 107) D198PDMKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTNETTNCTGGKNGIAADITTAKGYVKSVTTSNGAITVKGDGTLANMEYILQATGNAATGVTWTTTCKGTDASLFPANFCGSVTQ (SEQ ID NO. 108) D195PDMKLTTLQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTGKPSTCSGGNNGIAADIKTAKGYVASVKTQSGGITVKGDGTLANMEYILQATGNAATGVTWTTTCKGTDASLFPANFCGSVTQ (SEQ ID NO. 109) D195CDMKLTTLQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTGKPSTCSGGNNGIAADIKTAKGYVASVKTQSGGITVKGDGTLANMEYILQATGNAATGVTWTTTCKGTDASLFPANFCGSVTQ (SEQ ID NO. 110) D189PGMKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTNETTSCTGGKNGIAADITTAKGYVKSVTTSNGAITVAGNGTLDGMSYTLTAEGDSAKGVTWKTTCKGTDASLFPANFCGSVTQ (SEQ ID NO. 111) D189PDMKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTNETTSCTGGKNGIAADITTAKGYVKSVTTSNGAITVAGNGTLDGMSYTLTAEGDSAKGVTWKTTCKGTDASLFPANFCGSVTQ (SEQ ID NO. 112) D124PGMKLTTLQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTGKPSTCSGGNNGIAADIKTAKGYVASVKTQSGGITVKGDGTLANMEYILQATGNAATGVTWTTTCKGTDASLFPANFCGSVTQ (SEQ ID NO. 113) D124PDMKLTTLQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTGKPSTCSGGNNGIAADIKTAKGYVASVKTQSGGITVKGDGTLANMEYILQATGNAATGVTWTTTCKGTDASLFPANFCGSVTQ (SEQ ID NO. 114) D124CGMKLTTLQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTGKPSTCSGGNNGIAADIKTAKGYVASVKTQSGGITVKGDGTLANMEYILQATGNAATGVTWTTTCKGTDASLFPANFCGSVTQ (SEQ ID NO. 115) D58PGMKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTNETTNCTGGKNGIAADITTAKGYVASVKTQSGGITVKGDGTLANMEYILQATGNAATGVTWTTTCKGTEASLFPANFCGSVTQ (SEQ ID NO. 116) BS433MKLTTLQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTGKPSTCSGGNNGIAADIKTAKGYVASVKTQSGGITVKGDGTLANMEYILQATGNAATGVTWTTTCKGTDASLFPANFCGSVTQ (SEQ ID NO. 117) BS432MKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTGKPSTCSGGSNGIAADITTAKGYVASVKTQSGGITVKGNGTLANMEYILQAKGNATAGVTWTTTCKGTDASLFPANFCRSVTK (SEQ ID NO. 118) BS430MKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTNEATKCTGGKNGIAADITTAKGYVKSVTTSNGAITVKGDGTLANMEYILQASGNAATGVTWTTTCKGTDASLFPANFCGSVTQ (SEQ ID NO. 119) 1714MKLTTLQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTGKPSTCSGGSNGIAADITTAKGYVASVKTQSGGITVKGNGTLANMEYILQATGNAATGVTWTTTCKGTDASLFPANFCGSVTQ (SEQ ID NO. 120) 1128MKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKASVSELLQASAPYKSDVELCVYSTGKPSTCSGGSNGIAADITTAKGYVASVKTQSGGITVKGNGTLANMEYILQAKGNATAGVTWTTTCKGTDASLFPANFCRSVTK (SEQ ID NO. 121)

PilA may be PilA from H. influenzae strain NTHi3219C, NTHi3224A, NTHi12,NTHi44, NTHi67, 1054MEE, 1729MEE, 1728MEE, 1885MEE, 1060MEE, RdKW20,214NP, 1236MEE, 1714MEE, 1128MEE, 86-028NP, R2846, R2866, 3655, PittAA,PittGG, PittII, R3021, 22.4-21, 3185A, 3221B, 3241A, 038144S1, 821246,840645, 902550Z19, A840177, A920030, A950014, 901905U, A920029, A930105,306543X4, N218, N163, N162, N120, N107, N92, N91, D219PG, D211PG,D211PD, D204CD, D198PG, D198PD, D195PD, D195CD, D189PG, D189PD, D124PG,D124PD, D124CG, D58PG, BS433, BS432, BS430, 1714 or 1128. An amino acidsequence for PilA from H. influenzae strain D204CD is set forth in SEQID NO. 106, wherein X at position #116 is either glutamine (Q) orleucine (L); ambiguity as to the amino acid at position #116 could becleared up by technical resolution of the second nucleotide encodingamino acid #116, clarifying the PilA sequence for strain D204CD. PilAmay be PilA as set forth in any of SEQ ID NO. 58-SEQ ID NO. 121.

PilA may be a sequence with at least 95% identity, over the entirelength, to any of SEQ ID NO. 58-SEQ ID NO. 121 (as set out in Table 2).

Immunogenic fragments of PilA comprise immunogenic fragments of at least7, 10, 15, 20, 25, 30 or 50 contiguous amino acids of SEQ ID NO. 58-SEQID NO. 121. The immunogenic fragments may elicit antibodies which canbind the full length sequence from which the fragment is derived.

For example, immunogenic fragments of PilA comprise immunogenicfragments of at least 7, 10, 15, 20, 25, 30 or 50 contiguous amino acidsof SEQ ID NO. 58. The immunogenic fragments may elicit antibodies whichcan bind SEQ ID NO. 58.

Identity between polypeptides may be calculated by various algorithms.For example, the Needle program, from the EMBOSS package (Free software;EMBOSS: The European Molecular Biology Open Software Suite (2000).Trends in Genetics 16(6): 276-277) and the Gap program from the GCG®package (Accelrys Inc.) may be used. This Gap program is animplementation of the Needleman-Wunsch algorithm described in:Needleman, S. B. and Wunsch, C. D. (1970) J. Mol. Biol. 48, 443-453. TheBLOSUM62 scoring matrix has been used, and the gap open and extensionpenalties were respectively 8 and 2.

Looking at the computed alignment, identical residues between twocompared sequences can be observed. A percentage of identity can becomputed by (1) calculating the number of identities divided by thelength of the alignment, multiplied by 100 (for example, for the Needleprogram analysis), (2) calculating the number of identities divided bythe length of the longest sequence, multiplied by 100, (3) calculatingthe number of identities divided by the length of the shortest sequence,multiplied by 100, or (4) calculating the number of identities dividedby the number of aligned residues, multiplied by 100 (a residue isaligned if it is in front of another) (for example, for the Gap programanalysis).

As used herein, “adjuvant” means a compound or substance that, whenadministered to a subject in conjunction with a vaccine,immunotherapeutic, or other antigen- or immunogen-containingcomposition, increases or enhances the subject's immune response to theadministered antigen or immunogen (as compared to the immune responsethat would be obtained in the absence of adjuvant). This is to bedistinguished from “adjuvant therapy”, defined by the National CancerInstitute of the United States Institutes of Health in the context ofcancer treatment as additional treatment given after the primarytreatment, to lower the risk that the cancer will recur.

Conservative substitutions are well known and are generally set up asthe default scoring matrices in sequence alignment computer programs.These programs include PAM250 (Dayhoft M. O. et al., (1978), “A model ofevolutionary changes in proteins”, In “Atlas of Protein sequence andstructure” 5(3) M. O. Dayhoft (ed.), 345-352), National BiomedicalResearch Foundation, Washington, and Blosum 62 (Steven Henikoft andJorja G. Henikoft (1992), “Amino acid substitution matrices from proteinblocks”), Proc. Natl. Acad. Sci. USA 89 (Biochemistry): 10915-10919. Theinvention further provides fusion proteins of formula (I) containingconservative amino acid substitutions. For example, the fusion proteinsof formula (I) may contain a conservative substitution of any amino acidfrom PE or PilA of H. influenzae as described in any of the sequencesset forth herein (for example, any PE sequence set forth in SEQ ID NO.4-SEQ ID NO. 57 and/or any PilA sequence set forth in SEQ ID NO. 58-SEQID NO. 121)

As used herein “signal peptide” refers to a short (less than 60 aminoacids, for example, 3 to 60 amino acids) polypeptide present onprecursor proteins (typically at the N terminus), and which is typicallyabsent from the mature protein. The signal peptide (sp) is typicallyrich in hydrophobic amino acids. The signal peptide directs thetransport and/or secretion of the translated protein through themembrane. Signal peptides may also be called targeting signals, transitpeptides, localization signals, or signal sequences. For example, thesignal sequence may be a co-translational or post-translational signalpeptide.

A heterologous signal peptide may be cleaved from a fusion proteinconstruct by signal peptide peptidases during or after proteintransportation or secretion. For example, the signal peptide peptidaseis signal peptide peptidase I. A “heterologous” signal peptide is onewhich is not associated with the protein as it exists in nature.

As used herein “treatment” means the prevention of occurrence ofsymptoms of the condition or disease in a subject, the prevention ofrecurrence of symptoms of the condition or disease in a subject, thedelay of recurrence of symptoms of the condition or disease in asubject, the decrease in severity or frequency of symptoms of thecondition or disease in a subject, slowing or eliminating theprogression of the condition and the partial or total elimination ofsymptoms of the disease or condition in a subject.

As used herein, “optionally” means that the subsequently describedevent(s) may or may not occur, and includes both event(s) that occur andevents that do not occur.

The pathogenesis of disease caused by NTHi begins with nasopharyngealcolonization. Mechanisms to adhere to and maintain long-term residencewithin the nasopharyngeal micro-environment are considered ‘virulencedeterminants’ for NTHi. (Vaccine 28: 279-289 (2010)).

The importance of NTHi being able to adhere to the mucosal epithelialsurfaces of a human host is reflected in the multiplicity of adhesinsexpressed by NTHi. For example, some NTHi express pili. Other adhesivestructures belong to the autotransporter family of proteins; theseinclude Hap, HMW1/HMW2 and Hia/Hsf proteins. Further outer membraneproteins, such as the P2 protein, P5 protein and OapA have beendescribed as adhesions for Haemophilus influenzae. (CellularMicrobiology 4:191-200 (2002), Microbes and Infection 10: 87-96 (2008),Vaccine 28: 279-289 (2010)).

Otitis media is a major cause of morbidity in 80% of all children lessthan 3 years of age. (Expert Rev. Vaccines 5:517-534 (2006)). More than90% of children develop otitis media before age 7 (Current Opinion inInvestigational Drugs 4:953-958 (2003)). In 2000, there were 16 millionvisits made to office-based physicians for otitis media in the UnitedStates and approximately 13 million antibacterial prescriptionsdispensed. (Pediatrics 113:1451-1465 (2004)). In European countries, thereported acute otitis media rates range between 0.125 to 1.24 perchild-year. (Expert Review of Vaccines 8:1479-1500 (2009)). Otitis mediais a costly infection and the most common reason children receiveantibiotics. (Current Infectious Disease Reports 11:177-182 (2009)).Bacteria are responsible for approximately 70% of cases of acute otitismedia, with Streptococcus pneumoniae, non-typeable Haemophilusinfluenzae, and Moraxella catarrhalis predominating as the causativeagents (Expert Review of Vaccines 5:517-534 (2006)). A subset ofchildren experience recurrent and chronic otitis media and these otitisprone children have protracted middle-ear effusions that are associatedwith hearing loss and delays in speech and language development.(Current Infectious Disease Reports 11:177-182 (2009)).

Following the introduction of the heptavalent pneumococcal vaccine inmany countries, some studies have demonstrated a significant increase inthe proportion of acute otitis media caused by H. influenzae, with H.influenzae becoming the predominant pathogen. (Pediatric InfectiousDisease Journal 23:824-828; Pediatric Infectious Disease Journal23:829-833 (2004)).

Since otitis media is a multifactorial disease, the feasibility ofpreventing otitis media using a vaccination strategy has beenquestioned. (Current Infectious Disease Reports 11:177-182 (2009)).However, the results from one study suggest that it is possible for anantigen to induce at least partial protection against non-typeable H.influenzae. (Lancet 367:740-748 (2006)). One approach to developingvaccine antigens is to use antigenically conserved regions ofgenetically heterogeneous but abundantly expressed surface molecules.Another approach is to identify surface proteins that demonstratesequence or functional epitope conservation. A third consideration for avaccine antigen could be to select an antigen that is expressed duringinfection and colonization in a human host. Murphy (Curr. Infect.Disease Reports 11:177-182 (2009) states that, despite the existence ofseveral potential non-typeable H. influenzae candidate antigens, onecannot predict with certainty whether the candidate antigen will beeffective. (Current Infectious Disease Reports 11:177-182 (2009)). Someof the proteins described as potential vaccine antigens are: Haemophilusadhesin protein (Hap), High molecular-weight (HMW) proteins 1 and 2, H.influnzae adhesin (Hia), D15 protein, HtrA heat shock protein, P2surface protein, lipoprotein D, P5 fimbrin derived peptides, outermembrane protein P4, outer membrane protein (OMP) 26 (OMP26), P6protein, Protein E, Type IV pilus, lipooligosaccharide and phosphorylcholine. (Current Infectious Disease Reports 11:177-182 (2009); ExpertReview of Vaccines 5:517-534 (2006)).

The chinchilla model is a robust and validated animal model of otitismedia and its prevention (Expert Review of Vaccines 8:1063-1082 (2009)).While the chinchilla model may mimic the natural course of humaninfection, others have suggested that results in the chinchilla modelmay vary from one laboratory to the next. (Current Opinion inInvestigational Drugs 4:953-958 (2003)).

Various other rodents have also been used for the induction of otitismedia and are summarized in Vaccine 26:1501-1524 (2008). The murineanimal model is often studied in otitis media research.

The presence of bactericidal antibody is associated with protection fromotitis media due to non-typeable H. influenzae. (Current Opinion inInfectious Disease 16:129-134 (2003)). However, an immune response neednot be bactericidal to be effective against NTHi. Antibodies that merelyreact with NTHi surface adhesins can reduce or eliminate otitis media inthe chinchilla. (Current Opinion in Investigational Drugs 4:953-958(2003)).

Chronic obstructive pulmonary disease is a chronic inflammatory diseaseof the lungs and a major cause of morbidity and mortality worldwide.Approximately one in 20 deaths in 2005 in the US had COPD as theunderlying cause. (Drugs and Aging 26:985-999 (2009)). It is projectedthat in 2020 COPD will rise to the fifth leading cause of disabilityadjusted life years, chronic invalidating diseases, and to the thirdmost important cause of mortality (Lancet 349:1498-1504 (1997)).

The course of COPD is characterized by progressive worsening of airflowlimitation and a decline in pulmonary function. COPD may be complicatedby frequent and recurrent acute exacerbations (AE), which are associatedwith enormous health care expenditure and high morbidity. (Proceedingsof the American Thoracic Society 4:554-564 (2007)). One study suggeststhat approximately 50% of acute exacerbations of symptoms in COPD arecaused by non-typeable Haemophilus influenzae, Moraxella catarrhalis,Streptococcus pneumoniae, and Pseudomonas aeruginosa. (Drugs and Aging26:985-999 (2009)). H. influenzae is found in 20-30% of exacerbations ofCOPD; Streptococcus pneumoniae, in 10-15% of exacerbations of COPD; andMoraxella catarrhalis, in 10-15% of exacerbations of COPD. (New EnglandJournal of Medicine 359:2355-2365 (2008)). Haemophilus influenzae,Streptococcus pneumoniae, and Moraxella catarrhalis have been shown tobe the primary pathogens in acute exacerbations of bronchitis in HongKong, South Korea, and the Phillipines, while Klebsiella spp.,Pseudomonas aeruginosa and Acinetobacter spp. constitute a largeproportion of pathogens in other Asian countries/regions includingIndonesia, Thailand, Malaysia and Taiwan (Respirology, (2011) 16,532-539; doi:10.1111/j.1440.1843.2011.01943.x). In Bangladesh, 20% ofpatients with COPD showed positive sputum culture for Pseudomonas,Klebsiella, Streptococcus pneumoniae and Haemophilus influenzae, while65% of patients with AECOPD showed positive cultures for Pseudomonas,Klebsiella, Acinetobacter, Enterobacter, Moraxella catarrhalis andcombinations thereof. (Mymensingh Medical Journal 19:576-585 (2010)).However, it has been suggested that the two most important measures toprevent COPD exacerbation are active immunizations and chronicmaintenance of pharmacotherapy. (Proceedings of the American ThoracicSociety 4:554-564 (2007)).

There is a need for effective vaccines against NTHi. Using antigens thatmay act at different steps in pathogenesis may improve the efficacy of avaccine. The inventors have found that PilA and PE may be beneficiallypresent in the immunogenic compositions of the invention as fusionproteins.

The present invention relates to fusion proteins of formula (I).(X)_(m)—(R₁)_(n)-A-(Y)₀—B—(Z)_(p)  (formula I)

wherein:

X is a signal peptide or MHHHHHH (SEQ ID NO. 2);

m is 0 or 1;

R₁ is an amino acid;

n is 0, 1, 2, 3, 4, 5 or 6;

A is Protein E from Haemophilus influenzae or an immunogenic fragmentthereof, or PilA from Haemophilus influenzae or an immunogenic fragmentthereof;

Y is selected from the group consisting of GG, SG, SS and (G)_(h)wherein h is 4, 5, 6, 7, 8, 9, or 10;

o is 0 or 1;

B is PilA from Haemophilus influenzae or an immunogenic fragmentthereof, or Protein E from Haemophilus influenzae or an immunogenicfragment thereof;

Z is GGHHHHHH (SEQ ID NO: 3); and

p is 0 or 1.

In one embodiment, the fusion proteins of formula (I) are definedwherein X is selected from the group consisting of the signal sequencefrom CcmH (cytochrome c membrane protein H), DsbA (periplasmic proteindisulfide isomerise I), DsbB (disulfide bond membrane protein B), FlgI(flagellar peptidoglycan ring protein), FocC (F1c Chaperone protein),MalE (maltose transporter subunit E), NadA (quinolinate synthase subunitA), NikA (nickel ABC transporter component A), NspA (Neisserial surfaceprotein A), Omp26 (outer membrane protein 26), OmpA (outer membraneprotein A), OspA (outer surface protein A), pelB (pectate lyase B), PhoA(bacterial alkaline phosphatase), PhtD (pneumococcal histidine triadprotein D), PhtE (pneumococcal histidine triad protein E), SfmC(periiplasmic pilin chaperone), Sip1 (surface immunogenic protein), TolB(Tol-Pal Cell Envelope Complex Component B), TorA (trimethylamineN-oxide reductase system subunit A), TorT (trimethylamine N-oxidereductase system periplasmic protein T) and YraI (putative periplasmicpilin chaperone); or any subgroup thereof. In one embodiment, X is aco-translational signal peptide or a post-translational signal peptide.In one embodiment X is the signal sequence from FlgI (figI sp). Inanother particular embodiment, X is the signal sequence from pelB (pelBsp). In another embodiment, X is a post-translational signal peptide. Inanother embodiment, X is selected from the group consisting of thesignal sequence from FlgI, NadA and pelB.

In one embodiment, the fusion proteins of formula (I) are definedwherein m is 1. In another embodiment, m is 0.

In one particular embodiment, R₁ and n are defined wherein (R₁)_(n) is 1to 6 amino acids enriched in small, usually hydrophilic, amino acids.Hydrophilic amino acids include glutamic acid (E), aspartic acid (D) andasparagine (N).

In one embodiment, the fusion proteins of formula (I) are definedwherein n is selected from the group consisting of 0, 1, 2 and 6. In oneparticular embodiment, R₁ and n are defined wherein (R₁)_(n) is selectedfrom the group consisting of D, E, ATNDDD (SEQ ID NO. 178) and MD, orany subset thereof.

In one particular embodiment, n is selected from the group consisting of1, 2 and 6. In one particular embodiment, n is 0.

In one embodiment, the fusion proteins of formula (I) are definedwherein A is Protein E from H. influenzae. In another embodiment, thefusion proteins of formula (I) are defined wherein A is Protein E asencoded by an amino acid sequence selected from the group consisting ofSEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8,SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO.13, SEQ ID NO. 14, SEQ ID NO. 15, SEQ ID NO. 16, SEQ ID NO. 17, SEQ IDNO. 18, SEQ ID NO. 19, SEQ ID NO. 20, SEQ ID NO. 21, SEQ ID NO. 22, SEQID NO. 23, SEQ ID NO. 24, SEQ ID NO. 25, SEQ ID NO. 26, SEQ ID NO. 27,SEQ ID NO. 28, SEQ ID NO. 29, SEQ ID NO. 30, SEQ ID NO. 31, SEQ ID NO.32, SEQ ID NO. 33, SEQ ID NO. 34, SEQ ID NO. 35, SEQ ID NO. 36, SEQ IDNO. 37, SEQ ID NO. 38, SEQ ID NO. 39, SEQ ID NO. 40, SEQ ID NO. 41, SEQID NO. 42, SEQ ID NO. 43 SEQ ID NO. 44, SEQ ID NO. 45, SEQ ID NO. 46,SEQ ID NO. 47, SEQ ID NO. 48, SEQ ID NO. 49, SEQ ID NO. 50, SEQ ID NO.51, SEQ ID NO. 52, SEQ ID NO. 53, SEQ ID NO. 54, SEQ ID NO. 55, SEQ IDNO. 56 and SEQ ID NO. 57; or any subset of SEQ ID NO. 5 through SEQ IDNO. 57. In another embodiment, the fusion proteins of formula (I) aredefined wherein A is Protein E, wherein Protein E is approximately 75%to 100% identical to the Protein E amino acid sequence set forth in SEQID NO: 4. In another embodiment, A is Protein E wherein Protein E isapproximately 90% to 100% identical to the Protein E amino acid sequenceset forth in SEQ ID NO: 4. In another embodiment, A is Protein E whereinProtein E is at least 95% identical to the Protein E amino acid sequenceset forth in SEQ ID NO: 4. In additional embodiment, A is Protein Ewherein Protein E is at least 95% identical to Protein E as set for inany of SEQ ID NO. 4-SEQ ID NO. 57. In a particular embodiment, A isProtein E having the amino acid sequence set forth in SEQ ID NO. 4.

In another embodiment, the fusion proteins of formula (I) are definedwherein A is an immunogenic fragment of Protein E from H. influenzae. Inanother embodiment, A is an immunogenic fragment of Protein E whereinProtein E has an amino acid sequence selected from the group consistingof SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8,SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO.13, SEQ ID NO. 14, SEQ ID NO. 15, SEQ ID NO. 16, SEQ ID NO. 17, SEQ IDNO. 18, SEQ ID NO. 19, SEQ ID NO. 20, SEQ ID NO. 21, SEQ ID NO. 22, SEQID NO. 23, SEQ ID NO. 24, SEQ ID NO. 25, SEQ ID NO. 26, SEQ ID NO. 27,SEQ ID NO. 28, SEQ ID NO. 29, SEQ ID NO. 30, SEQ ID NO. 31, SEQ ID NO.32, SEQ ID NO. 33, SEQ ID NO. 34, SEQ ID NO. 35, SEQ ID NO. 36, SEQ IDNO. 37, SEQ ID NO. 38, SEQ ID NO. 39, SEQ ID NO. 40, SEQ ID NO. 41, SEQID NO. 42, SEQ ID NO. 43 SEQ ID NO. 44, SEQ ID NO. 45, SEQ ID NO. 46,SEQ ID NO. 47, SEQ ID NO. 48, SEQ ID NO. 49, SEQ ID NO. 50, SEQ ID NO.51, SEQ ID NO. 52, SEQ ID NO. 53, SEQ ID NO. 54, SEQ ID NO. 55, SEQ IDNO. 56 and SEQ ID NO. 57; or any subset of SEQ ID NO. 4 through SEQ IDNO. 57. In another embodiment, A is an immunogenic fragment of ProteinE, wherein Protein E is approximately 75% to 100% identical to the aminoacid sequence set forth in SEQ ID NO: 4. In another embodiment, A is animmunogenic fragment of Protein E, wherein Protein E is approximately90% to 100% identical to SEQ ID NO. 4. In an additional embodiment, A isan immunogenic fragment of Protein E, wherein Protein E is at least 95%identical to any of SEQ ID NO. 4-SEQ ID NO. 57. More specifically, inone embodiment, A is an immunogenic fragment of Protein E, whereinProtein E is 93% to 100% identical to SEQ ID NO. 124. In a particularembodiment, A is an immunogenic fragment of Protein E wherein Protein Eis SEQ ID NO. 4.

In another embodiment, A is an immunogenic fragment of Protein E from H.influenzae selected from the group consisting of amino acids 17-160 ofSEQ ID NO. 4 (SEQ ID NO. 122), amino acids 18-160 of SEQ ID NO. 4 (SEQID NO. 123), amino acids 19-160 of SEQ ID NO. 4 (SEQ ID NO. 124), aminoacids 20-160 of SEQ ID NO. 4 (SEQ ID NO. 125) and amino acids 22-160 ofSEQ ID NO. 4 (SEQ ID NO. 126). In another embodiment, A is animmunogenic fragment of Protein E from H. influenzae selected from thegroup consisting of amino acids 17-160 of SEQ ID NO. 4 (SEQ ID NO. 122),amino acids 18-160 of SEQ ID NO. 4 (SEQ ID NO. 123), amino acids 19-160of SEQ ID NO. 4 (SEQ ID NO. 124), amino acids 20-160 of SEQ ID NO. 4(SEQ ID NO. 125), amino acids 22-160 of SEQ ID NO. 4 (SEQ ID NO. 126),amino acids 23-160 of SEQ ID NO. 4 (SEQ ID NO. 179) and amino acids24-160 of SEQ ID NO. 4 (SEQ ID NO. 180). In a further embodiment, A isan immunogenic fragment of Protein E from H. influenzae selected fromthe group consisting of amino acids 17-160 of SEQ ID NO. 4 (SEQ ID NO.122), amino acids 18-160 of SEQ ID NO. 4 (SEQ ID NO. 123), amino acids20-160 of SEQ ID NO. 4 (SEQ ID NO. 125), amino acids 22-160 of SEQ IDNO. 4 (SEQ ID NO. 126), amino acids 23-160 of SEQ ID NO. 4 (SEQ ID NO.179) and amino acids 24-160 of SEQ ID NO. 4 (SEQ ID NO. 180). Morespecifically, in one embodiment, A is SEQ ID NO. 124, amino acids 19-160of SEQ ID NO. 4. In an additional embodiment, A is SEQ ID NO. 125, aminoacids 20-160 of SEQ ID NO. 5. In another embodiment, A is immunogenicfragment of Protein E from H. influenzae selected from the groupconsisting of amino acids 23-160 of SEQ ID NO. 4 (SEQ ID NO. 179) andamino acids 24-160 of SEQ ID NO. 4 (SEQ ID NO. 180).

Protein E- SEQ ID NO. 4MKKIILTLSL GLLTACSAQI QKAEQNDVKL APPTDVRSGY IRLVKNVNYYIDSESIWVDN QEPQIVHFDA VVNLDKGLYV YPEPKRYARS VRQYKILNCANYHLTQVRTD FYDEFWGQGL RAAPKKQKKH TLSLTPDTTL YNAAQIICAN YGEAFSVDKKAmino acids 17-160 of Protein E from SEQ ID NO. 4- SEQ ID NO. 122                 SAQI QKAEQNDVKL APPTDVRSGY IRLVKNVNYYIDSESIWVDN QEPQIVHFDA VVNLDKGLYV YPEPKRYARS VRQYKILNCANYHLTQVRTD FYDEFWGQGL RAAPKKQKKH TLSLTPDTTL YNAAQIICAN YGEAFSVDKKAmino acids 18-160 of Protein E from SEQ ID NO. 4- SEQ ID NO. 123                  AQI QKAEQNDVKL APPTDVRSGY IRLVKNVNYYIDSESIWVDN QEPQIVHFDA VVNLDKGLYV YPEPKRYARS VRQYKILNCANYHLTQVRTD FYDEFWGQGL RAAPKKQKKH TLSLTPDTTL YNAAQIICAN YGEAFSVDKKAmino acids 19-160 of Protein E from SEQ ID NO. 4- SEQ ID NO. 124                   QI QKAEQNDVKL APPTDVRSGY IRLVKNVNYYIDSESIWVDN QEPQIVHFDA VVNLDKGLYV YPEPKRYARS VRQYKILNCANYHLTQVRTD FYDEFWGQGL RAAPKKQKKH TLSLTPDTTL YNAAQIICAN YGEAFSVDKKAmino acids 20-160 of Protein E from SEQ ID NO. 4- SEQ ID NO. 125                    I QKAEQNDVKL APPTDVRSGY IRLVKNVNYYIDSESIWVDN QEPQIVHFDA VVNLDKGLYV YPEPKRYARS VRQYKILNCANYHLTQVRTD FYDEFWGQGL RAAPKKQKKH TLSLTPDTTL YNAAQIICAN YGEAFSVDKKAmino acids 22-160 of Protein E from SEQ ID NO. 4- SEQ ID NO. 126                       KAEQNDVKL APPTDVRSGY IRLVKNVNYYIDSESIWVDN QEPQIVHFDA VVNLDKGLYV YPEPKRYARS VRQYKILNCANYHLTQVRTD FYDEFWGQGL RAAPKKQKKH TLSLTPDTTL YNAAQIICAN YGEAFSVDKKAmino acids 23-160 of Protein E from SEQ ID NO. 4- SEQ ID NO. 179                        AEQNDVKL APPTDVRSGY IRLVKNVNYYIDSESIWVDN QEPQIVHFDA VVNLDKGLYV YPEPKRYARS VRQYKILNCANYHLTQVRTD FYDEFWGQGL RAAPKKQKKH TLSLTPDTTL YNAAQIICAN YGEAFSVDKKAmino acids 24-160 Protein E from SEQ ID NO.- SEQ ID NO. 180                         EQNDVKL APPTDVRSGY IRLVKNVNYYIDSESIWVDN QEPQIVHFDA VVNLDKGLYV YPEPKRYARS VRQYKILNCANYHLTQVRTD FYDEFWGQGL RAAPKKQKKH TLSLTPDTTL YNAAQIICAN YGEAFSVDKK

In another embodiment, the fusion proteins of formula (I) are definedwherein A is PilA from H. influenzae. In another embodiment, the fusionproteins of formula (I) are defined wherein A is PilA from H. influenzaehaving an amino acid sequence selected from the group consisting of SEQID NO. 58, SEQ ID NO. 59, SEQ ID NO. 60, SEQ ID NO. 61, SEQ ID NO. 62,SEQ ID NO. 63, SEQ ID NO. 64, SEQ ID NO. 65, SEQ ID NO. 66, SEQ ID NO.67, SEQ ID NO. 68, SEQ ID NO. 69, SEQ ID NO. 70, SEQ ID NO. 71, SEQ IDNO. 72, SEQ ID NO. 73, SEQ ID NO. 74, SEQ ID NO. 75, SEQ ID NO. 76, SEQID NO. 77, SEQ ID NO. 78, SEQ ID NO. 79, SEQ ID NO. 80, SEQ ID NO. 81,SEQ ID NO. 82, SEQ ID NO. 83, SEQ ID NO. 84, SEQ ID NO. 85, SEQ ID NO.86, SEQ ID NO. 87, SEQ ID NO. 88, SEQ ID NO. 89, SEQ ID NO. 90, SEQ IDNO. 91, SEQ ID NO. 92, SEQ ID NO. 93, SEQ ID NO. 94, SEQ ID NO. 95, SEQID NO. 96, SEQ ID NO. 97, SEQ ID NO. 98, SEQ ID NO. 99, SEQ ID NO. 100,SEQ ID NO. 101, SEQ ID NO. 102, SEQ ID NO. 103, SEQ ID NO. 104, SEQ IDNO. 105, SEQ ID NO. 106, SEQ ID NO. 107, SEQ ID NO. 108, SEQ ID NO. 109,SEQ ID NO. 110, SEQ ID NO. 111, SEQ ID NO. 112, SEQ ID NO. 113, SEQ IDNO. 114, SEQ ID NO. 115, SEQ ID NO. 116, SEQ ID NO. 117, SEQ ID NO. 118,SEQ ID NO. 119, SEQ ID NO. 120 and SEQ ID NO. 121; or any subset of SEQID NO. 58 through SEQ ID NO. 121. In another embodiment, A is PilAwherein PilA is approximately 80% to 100% identical to SEQ ID NO. 58. Inanother embodiment, A is PilA wherein PilA is at least 95% identical toany of SEQ ID NO. 58-SEQ ID NO. 121. In a particular embodiment, A isPilA of SEQ ID NO. 58.

In another embodiment, the fusion proteins of formula (I) are definedwherein A an immunogenic fragment of PilA from H. influenzae. In anotherembodiment, A is an immunogenic fragment of PilA wherein PilA isapproximately 80% to 100% identical to SEQ ID NO. 58. For example, A isan immunogenic fragment of PilA wherein PilA has an amino acid sequenceselected from the group consisting of SEQ ID NO. 58, SEQ ID NO. 59, SEQID NO. 60, SEQ ID NO. 61, SEQ ID NO. 62, SEQ ID NO. 63, SEQ ID NO. 64,SEQ ID NO. 65, SEQ ID NO. 66, SEQ ID NO. 67, SEQ ID NO. 68, SEQ ID NO.69, SEQ ID NO. 70, SEQ ID NO. 71, SEQ ID NO. 72, SEQ ID NO. 73, SEQ IDNO. 74, SEQ ID NO. 75, SEQ ID NO. 76, SEQ ID NO. 77, SEQ ID NO. 78, SEQID NO. 79, SEQ ID NO. 80, SEQ ID NO. 81, SEQ ID NO. 82, SEQ ID NO. 83,SEQ ID NO. 84, SEQ ID NO. 85, SEQ ID NO. 86, SEQ ID NO. 87, SEQ ID NO.88, SEQ ID NO. 89, SEQ ID NO. 90, SEQ ID NO. 91, SEQ ID NO. 92, SEQ IDNO. 93, SEQ ID NO. 94, SEQ ID NO. 95, SEQ ID NO. 96, SEQ ID NO. 97, SEQID NO. 98, SEQ ID NO. 99, SEQ ID NO. 100, SEQ ID NO. 101, SEQ ID NO.102, SEQ ID NO. 103, SEQ ID NO. 104, SEQ ID NO. 105, SEQ ID NO. 106, SEQID NO. 107, SEQ ID NO. 108, SEQ ID NO. 109, SEQ ID NO. 110, SEQ ID NO.111, SEQ ID NO. 112, SEQ ID NO. 113, SEQ ID NO. 114, SEQ ID NO. 115, SEQID NO. 116, SEQ ID NO. 117, SEQ ID NO. 118, SEQ ID NO. 119, SEQ ID NO.120 and SEQ ID NO. 121; or any subset SEQ ID NO. 58 through SEQ ID NO.121. In an additional embodiment, A is an immunogenic fragment of PilAwherein PilA is at least 95% identical to any of SEQ ID NO. 58-SEQ IDNO. 121. In a particular embodiment, A is an immunogenic fragment ofPilA from H. influenzae strain 86-028NP wherein PilA is SEQ ID NO. 58.

PilA from H. influenzae strain 86-028NP SEQ ID NO. 58MKLTTQQTLK KGFTLIELMI VIAIIAILAT IAIPSYQNYTKKAAVSELLQ ASAPYKADVE LCVYSTNETT NCTGGKNGIAADITTAKGYV KSVTTSNGAI TVKGDGTLAN MEYILQATGNAATGVTWTTT CKGTDASLFP ANFCGSVTQ

In another embodiment, A is an immunogenic fragment of PilAapproximately 75% to 100% identical to SEQ ID NO. 127. Morespecifically, in one embodiment A is SEQ ID NO. 127, a fragmentconsisting of amino acids 40-149 of SEQ ID NO. 58.

Amino acids 40-149 of PilA from H. influenzae strain 86-028NPSEQ ID NO. 127. T KKAAVSELLQ ASAPYKADVE LCVYSTNETT NCTGGKNGIAADITTAKGYV KSVTTSNGAI TVKGDGTLAN MEYILQATGNAATGVTWTTT CKGTDASLFP ANFCGSVTQ

In another embodiment, A is an immunogenic fragment of PilA consistingof amino acids 40-149 from any of SEQ ID NO. 58-SEQ ID NO. 121. In anadditional embodiment, A is an immunogenic fragment at least 95%identical to amino acids 40-149 from any of SEQ ID NO. 58-SEQ ID NO.121.

In one embodiment, the fusion proteins of formula (I) are definedwherein Y is selected from the group consisting of GG, SG and SS. Inanother embodiment, the fusion proteins of formula (I) are definedwherein Y is GG or SG. In one particular embodiment, Y is GG.

In one embodiment, the fusion proteins of formula (I) are definedwherein o is 1. In another embodiment, o is 0.

In one embodiment, the fusion proteins of formula (I) are definedwherein B is PilA from H. influenzae or an immunogenic fragment of PilAfrom H. influenzae when A is Protein E from H. influenzae or animmunogenic fragment of Protein E from H. influenzae. For example, B isPilA from H. influenzae strain 86-028NP. In another embodiment, B isPilA from H. influenzae having an amino acid sequence selected from thegroup consisting of SEQ ID NO. 58, SEQ ID NO. 59, SEQ ID NO. 60, SEQ IDNO. 61, SEQ ID NO. 62, SEQ ID NO. 63, SEQ ID NO. 64, SEQ ID NO. 65, SEQID NO. 66, SEQ ID NO. 67, SEQ ID NO. 68, SEQ ID NO. 69, SEQ ID NO. 70,SEQ ID NO. 71, SEQ ID NO. 72, SEQ ID NO. 73, SEQ ID NO. 74, SEQ ID NO.75, SEQ ID NO. 76, SEQ ID NO. 77, SEQ ID NO. 78, SEQ ID NO. 79, SEQ IDNO. 80, SEQ ID NO. 81, SEQ ID NO. 82, SEQ ID NO. 83, SEQ ID NO. 84, SEQID NO. 85, SEQ ID NO. 86, SEQ ID NO. 87, SEQ ID NO. 88, SEQ ID NO. 89,SEQ ID NO. 90, SEQ ID NO. 91, SEQ ID NO. 92, SEQ ID NO. 93, SEQ ID NO.94, SEQ ID NO. 95, SEQ ID NO. 96, SEQ ID NO. 97, SEQ ID NO. 98, SEQ IDNO. 99, SEQ ID NO. 100, SEQ ID NO. 101, SEQ ID NO. 102, SEQ ID NO. 103,SEQ ID NO. 104, SEQ ID NO. 105, SEQ ID NO. 106, SEQ ID NO. 107, SEQ IDNO. 108, SEQ ID NO. 109, SEQ ID NO. 110, SEQ ID NO. 111, SEQ ID NO. 112,SEQ ID NO. 113, SEQ ID NO. 114, SEQ ID NO. 115, SEQ ID NO. 116, SEQ IDNO. 117, SEQ ID NO. 118, SEQ ID NO. 119, SEQ ID NO. 120 and SEQ ID NO.121; or any subset of SEQ ID NO. 58 through SEQ ID NO. 121. In anotherembodiment, B is PilA wherein PilA is approximately 80% to 100%identical to SEQ ID NO. 58. In another embodiment, B is PilA whereinPilA is at least 95% identical to any of SEQ ID NO. 58-SEQ ID NO. 121.In a particular embodiment, B is PilA of SEQ ID NO. 58.

In another embodiment, B is PilA wherein PilA is at least 95% identicalto any of SEQ ID NO. 58-SEQ ID NO. 121 and A is PE wherein PE is atleast 95% identical to any of SEQ ID NO. 4-SEQ ID NO. 57.

In another embodiment, the fusion proteins of formula (I) are definedwherein B is an immunogenic fragment of PilA from H. influenzae when Ais an immunogenic fragment of Protein E from H. influenzae. For example,B is an immunogenic fragment of the PilA from H. influenzae strain86-028NP. In another embodiment, B is an immunogenic fragment of PilAwherein PilA is approximately 80% to 100% identical to SEQ ID NO: 58. Inanother embodiment, B is an immunogenic fragment of PilA wherein PilAhas an amino acid selected from the group consisting of SEQ ID NO. 58,SEQ ID NO. 59, SEQ ID NO. 60, SEQ ID NO. 61, SEQ ID NO. 62, SEQ ID NO.63, SEQ ID NO. 64, SEQ ID NO. 65, SEQ ID NO. 66, SEQ ID NO. 67, SEQ IDNO. 68, SEQ ID NO. 69, SEQ ID NO. 70, SEQ ID NO. 71, SEQ ID NO. 72, SEQID NO. 73, SEQ ID NO. 74, SEQ ID NO. 75, SEQ ID NO. 76, SEQ ID NO. 77,SEQ ID NO. 78, SEQ ID NO. 79, SEQ ID NO. 80, SEQ ID NO. 81, SEQ ID NO.82, SEQ ID NO. 83, SEQ ID NO. 84, SEQ ID NO. 85, SEQ ID NO. 86, SEQ IDNO. 87, SEQ ID NO. 88, SEQ ID NO. 89, SEQ ID NO. 90, SEQ ID NO. 91, SEQID NO. 92, SEQ ID NO. 93, SEQ ID NO. 94, SEQ ID NO. 95, SEQ ID NO. 96,SEQ ID NO. 97, SEQ ID NO. 98, SEQ ID NO. 99, SEQ ID NO. 100, SEQ ID NO.101, SEQ ID NO. 102, SEQ ID NO. 103, SEQ ID NO. 104, SEQ ID NO. 105, SEQID NO. 106, SEQ ID NO. 107, SEQ ID NO. 108, SEQ ID NO. 109, SEQ ID NO.110, SEQ ID NO. 111, SEQ ID NO. 112, SEQ ID NO. 113, SEQ ID NO. 114, SEQID NO. 115, SEQ ID NO. 116, SEQ ID NO. 117, SEQ ID NO. 118, SEQ ID NO.119, SEQ ID NO. 120 and SEQ ID NO. 121; or any subset of SEQ ID NO. 58through SEQ ID NO. 121. In another embodiment, B is an immunogenicfragment of PilA wherein PilA is at least 95% identical to any of SEQ IDNO. 58-SEQ ID NO. 121. In a particular embodiment, B is an immunogenicfragment of PilA from H. influenzae wherein PilA has the amino acidsequence set forth in SEQ ID NO. 58. In another embodiment, B is animmunogenic fragment of PilA consisting of amino acids 40-149 from anyof SEQ ID NO. 58-SEQ ID NO. 121. More specifically, in one embodiment Bis the fragment of PilA as set forth in SEQ ID NO. 127. In an additionalembodiment, B is an immunogenic fragment at least 95% identical to aminoacids 40-149 of any of SEQ ID NO. 58-SEQ ID NO. 121.

In one particular embodiment, B is the fragment of PilA as set forth inSEQ ID NO. 127 and A is an immunogenic fragment of Protein E selectedfrom the group consisting of SEQ ID NO. 122, SEQ ID NO. 124, SEQ ID NO.125 and SEQ ID NO. 126. More particularly, B is the fragment of PilA asset forth in SEQ ID NO. 127 and A is the fragment of Protein E as setforth in SEQ ID NO. 124, amino acids 19-160 of Protein E from SEQ ID NO.4. In another embodiment, B is the fragment of PilA as set forth in SEQID NO. 127 and A is the fragment of Protein E as set forth in SEQ ID NO.125.

In another embodiment, B is an immunogenic fragment of PilA wherein PilAis at least 95% identical to any of SEQ ID NO. 58-SEQ ID NO. 121 and Ais an immunogenic fragment of PE wherein PE is at least 95% identical toany of SEQ ID NO. 4-SEQ ID NO. 57.

In another embodiment, the fusion proteins of formula (I) are definedwherein B is Protein E from H. influenzae when A is PilA from H.influenzae. For example, B is Protein E having an amino acid sequenceselected from the group consisting of SEQ ID NO. 4, SEQ ID NO. 5, SEQ IDNO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ IDNO. 11, SEQ ID NO. 12, SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 15, SEQID NO. 16, SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 19, SEQ ID NO. 20,SEQ ID NO. 21, SEQ ID NO. 22, SEQ ID NO. 23, SEQ ID NO. 24, SEQ ID NO.25, SEQ ID NO. 26, SEQ ID NO. 27, SEQ ID NO. 28, SEQ ID NO. 29, SEQ IDNO. 30, SEQ ID NO. 31, SEQ ID NO. 32, SEQ ID NO. 33, SEQ ID NO. 34, SEQID NO. 35, SEQ ID NO. 36, SEQ ID NO. 37, SEQ ID NO. 38, SEQ ID NO. 39,SEQ ID NO. 40, SEQ ID NO. 41, SEQ ID NO. 42, SEQ ID NO. 43 SEQ ID NO.44, SEQ ID NO. 45, SEQ ID NO. 46, SEQ ID NO. 47, SEQ ID NO. 48, SEQ IDNO. 49, SEQ ID NO. 50, SEQ ID NO. 51, SEQ ID NO. 52, SEQ ID NO. 53, SEQID NO. 54, SEQ ID NO. 55, SEQ ID NO. 56 and SEQ ID NO. 57; or any subsetof SEQ ID NO. 4 through SEQ ID NO. 57. In another embodiment, the fusionproteins of formula (I) are defined wherein B is Protein E whereinProtein E is approximately 75% to 100% identical to the Protein E aminoacid sequence set forth in SEQ ID NO: 4. In another embodiment, B isProtein E wherein Protein E is approximately 90% to 100% identical tothe Protein E amino acid sequence set forth in SEQ ID NO: 4. Forexample, B is Protein E wherein Protein E is at least 95% identical toProtein E as set forth in SEQ ID NO. 4. In another embodiment, B isProtein E wherein Protein E is at least 95% identical to any of SEQ IDNO. 4-SEQ ID NO. 57. In a particular embodiment, B is Protein E havingthe amino acid sequence set forth in SEQ ID NO. 4.

In another embodiment, the fusion proteins of formula (I) are definedwherein B is an immunogenic fragment of Protein E from H. influenzaewhen A is an immunogenic fragment of PilA from H. influenzae. Forexample, B is an immunogenic fragment of Protein E wherein Protein E hasan amino acid sequence selected from the group consisting of SEQ ID NO.4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9,SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13, SEQ ID NO.14, SEQ ID NO. 15, SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO. 18, SEQ IDNO. 19, SEQ ID NO. 20, SEQ ID NO. 21, SEQ ID NO. 22, SEQ ID NO. 23, SEQID NO. 24, SEQ ID NO. 25, SEQ ID NO. 26, SEQ ID NO. 27, SEQ ID NO. 28,SEQ ID NO. 29, SEQ ID NO. 30, SEQ ID NO. 31, SEQ ID NO. 32, SEQ ID NO.33, SEQ ID NO. 34, SEQ ID NO. 35, SEQ ID NO. 36, SEQ ID NO. 37, SEQ IDNO. 38, SEQ ID NO. 39, SEQ ID NO. 40, SEQ ID NO. 41, SEQ ID NO. 42, SEQID NO. 43, SEQ ID NO. 44, SEQ ID NO. 45, SEQ ID NO. 46, SEQ ID NO. 47,SEQ ID NO. 48, SEQ ID NO. 49, SEQ ID NO. 50, SEQ ID NO. 51, SEQ ID NO.52, SEQ ID NO. 53, SEQ ID NO. 54, SEQ ID NO. 55, SEQ ID NO. 56 and SEQID NO. 57; or any subset of SEQ ID NO. 4 through SEQ ID NO. 57. Inanother embodiment, the fusion proteins of formula (I) are definedwherein B is an immunogenic fragment of Protein E wherein Protein E isapproximately 75% to 100% identical to the Protein E amino acid sequenceset forth in SEQ ID NO. 4. In another embodiment, B is an immunogenicfragment of Protein E wherein Protein E is approximately 90% to 100%identical to the Protein E amino acid sequence set forth in SEQ ID NO:4. In a particular embodiment, B is an immunogenic fragment of Protein Ehaving the amino acid sequence set forth in SEQ ID NO. 4. In anadditional embodiment, B is an immunogenic fragment of Protein E,wherein Protein E is at least 95% identical to any of SEQ ID NO. 4-SEQID NO. 57.

In another embodiment, B is a fragment of Protein E from H. influenzaeselected from the group consisting of amino acids 17-160 of SEQ ID NO. 4(SEQ ID NO. 122), amino acids 18-160 of SEQ ID NO. 4 (SEQ ID NO. 123),amino acids 19-160 of SEQ ID NO. 4 (SEQ ID NO. 124), amino acids 20-160of SEQ ID NO. 4 (SEQ ID NO. 125) and amino acids 22-160 of SEQ ID NO. 4(SEQ ID NO. 126). In another embodiment, B is an immunogenic fragment ofProtein E from H. influenzae selected from the group consisting of aminoacids 17-160 of SEQ ID NO. 4 (SEQ ID NO. 122), amino acids 18-160 of SEQID NO. 4 (SEQ ID NO. 123), amino acids 19-160 of SEQ ID NO. 4 (SEQ IDNO. 124), amino acids 20-160 of SEQ ID NO. 4 (SEQ ID NO. 125), aminoacids 22-160 of SEQ ID NO. 4 (SEQ ID NO. 126), amino acids 23-160 of SEQID NO. 4 (SEQ ID NO. 179) and amino acids 24-160 of SEQ ID NO. 4 (SEQ IDNO. 180). More specifically, in one embodiment, B is the fragment ofProtein E as set forth in SEQ ID NO. 123, amino acids 18-160 of SEQ IDNO. 4.

In one particular embodiment B is an immunogenic fragment of Protein Eas set forth in SEQ ID NO. 123, amino acids 18-160 of SEQ ID NO. 4 whenA is an immunogenic fragment of PilA as set forth in SEQ ID NO. 127.

In one embodiment, the fusion proteins of formula (I) are definedwherein p is 0. In another embodiment, the fusion proteins of formula(I) are defined wherein p is 1.

In one embodiment, the fusion protein of formula (I) is selected fromthe group consisting of SEQ ID NO. 136, SEQ ID NO. 138, SEQ ID NO. 140,SEQ ID NO. 142, SEQ ID NO. 144, SEQ ID NO. 146, SEQ ID NO. 148, SEQ IDNO. 150, SEQ ID NO. 182, SEQ ID NO. 184, SEQ ID NO. 186, SEQ ID NO. 188,SEQ ID NO. 190, SEQ ID NO. 192, SEQ ID NO. 194, SEQ ID NO. 196, SEQ IDNO. 198, SEQ ID NO. 200, SEQ ID NO. 202 and SEQ ID NO. 204; or anysubset thereof. In another embodiment, the fusion protein of formula (I)is approximately 95% identical to any of SEQ ID NO. 136, SEQ ID NO. 138,SEQ ID NO. 140, SEQ ID NO. 142, SEQ ID NO. 144, SEQ ID NO. 146, SEQ IDNO. 148, SEQ ID NO. 150, SEQ ID NO. 182, SEQ ID NO. 184, SEQ ID NO. 186,SEQ ID NO. 188, SEQ ID NO. 190, SEQ ID NO. 192, SEQ ID NO. 194, SEQ IDNO. 196, SEQ ID NO. 198, SEQ ID NO. 200, SEQ ID NO. 202 or SEQ ID NO.204.

Fusion proteins of formula (I) are useful as immunogens in subjects suchas mammals, particularly humans. In particular, the fusion proteins offormula (I) are useful in inducing an immune response against H.influenzae in subjects, particularly humans. More specifically, thefusion proteins of formula (I) are useful in the treatment or preventionof otitis media and/or AECOPD and/or pneumonia.

The present invention relates to immunogenic compositions comprisingProtein E from H. influenzae (or an immunogenic fragment thereof) andPilA from H. influenzae (or an immunogenic fragment thereof), andimmunogenic compositions comprising fusion proteins of Protein E from H.influenzae (or an immunogenic fragment thereof) and PilA from H.influenzae (or an immunogenic fragment thereof). The present inventionalso relates to vaccines comprising such immunogenic compositions andtherapeutic uses of the same.

In one embodiment, the immunogenic compositions comprise Protein E fromH. influenzae (or an immunogenic fragment thereof) and PilA from H.influenzae (or an immunogenic fragment thereof). Protein E may be SEQ IDNO. 4 or a Protein E sequence at least 75%, 80%, 85%, 90%, 95%, 96%,97%, 98% or 99% identical to SEQ ID NO. 4.

The immunogenic fragment of Protein E may be SEQ ID NO. 122, SEQ ID NO.123, SEQ ID NO. 124, SEQ ID NO. 125 or SEQ ID NO. 126, or a sequencehaving at least 90%, 95%, 96%, 97%, 98%, 99% sequence identity to anyone of SEQ ID NO. 122, SEQ ID NO. 123, SEQ ID NO. 124, SEQ ID NO. 125 orSEQ ID NO. 126. The immunogenic fragment of Protein E may be SEQ ID NO.122, SEQ ID NO. 123, SEQ ID NO. 124, SEQ ID NO. 125, SEQ ID NO. 126, SEQID NO. 179 or SEQ ID NO. 180 or a sequence having at least 90%, 95%,96%, 97%, 98%, 99% sequence identity to any one of SEQ ID NO. 122, SEQID NO. 123, SEQ ID NO. 124, SEQ ID NO. 125, SEQ ID NO. 126, SEQ ID NO.179 or SEQ ID NO. 180. Amino acid differences have been described inProtein E from various Haemophilus species when compared to Protein Efrom Haemophilus influenzae Rd as a reference strain. Microbes &Infection (Corrigendum to “Identification of a novel Haemophilusinfluenzae protein important for adhesion to epithelia cells” [MicrobesInfect. 10 (2008) 87-97], available online Jul. 6, 2010, “Article inPress”) provides a sequence for Protein E from H. influenzae strain 772.WO2002/28889 provides a sequence for Protein E from H. influenzae strain12085.

Protein E contains an epithelial cell binding region (PKRYARSVRQYKILNCANYH LTQVR, SEQ ID NO. 128) that has been reported to be conservedamong more than 100 clinical NTHi isolates, encapsulated H. influenzae,and culture collection strains analyzed (Singh et al, J. Infect. Dis.201(3):414-9 (2010)). Singh et al. reported that Protein E was highlyconserved in both NTHi and encapsulated H. influenzae (96.9%-100%identity without the signal peptide). In one embodiment, the fragment ofProtein E comprises the binding region of SEQ ID NO. 128 (PKRYARSVRQYKILNCANYH LTQVR).

PilA is a conserved adhesin expressed in vivo. Full length comparison of64 sequences of PilA from Haemophilus influenzae demonstratedapproximately 80% to 100% identity.

In another embodiment, the immunogenic composition comprises a fusionprotein as defined by formula (I).

In one embodiment, the present immunogenic compositions may beadministered with other antigens from H. influenzae. For example, the PEand PilA or the fusion protein of formula (I) may be administered withProtein D from H. influenzae. Protein D may be as described inWO91/18926. In another embodiment, the immunogenic composition mayinclude the fusion protein of formula (I) and Protein D from H.influenzae.

In another embodiment, the immunogenic compositions of the invention maybe administered with additional antigens from other bacterial speciesalso known to cause otitis media, AECOPD or pneumonia.

The amount of the immunogenic composition which is required to achievethe desired therapeutic or biological effect will depend on a number offactors such as the use for which it is intended, the means ofadministration, the recipient and the type and severity of the conditionbeing treated, and will be ultimately at the discretion of the attendantphysician or veterinarian. In general, a typical dose for the treatmentof a condition caused in whole or in part by H. influenzae in a human,for instance, may be expected to lie in the range of from about 0.003 mgto about 0.090 mg. More specifically, a typical dose for the treatmentof a condition caused wholly or in part by H. influenzae in a human maylie in the range of from about 0.01 mg to about 0.03 mg of fusionprotein. The immunogenic composition may contain additional antigens; atypical dose for the treatment of a condition caused wholly or in partby H. influenzae in a human may lie in the range of from about 0.01 mgto about 0.03 mg for each additional antigen. This dose may beadministered as a single unit dose. Several separate unit doses may alsobe administered. For example, separate unit doses may be administered asseparate priming doses within the first year of life or as separatebooster doses given at regular intervals (for example, every 1, 5 or 10years).

Formulations comprising the immunogenic compositions of the inventionmay be adapted for administration by an appropriate route, for example,by the intramuscular, sublingual, transcutaneous, intradermal orintranasal route. Such formulations may be prepared by any method knownin the art.

The immunogenic compositions of the present invention may additionallycomprise an adjuvant. When the term “adjuvant” is used in thisspecification, it refers to a substance that is administered inconjunction with the immunogenic composition to boost the patient'simmune response to the immunogenic component of the composition.

Suitable adjuvants include an aluminum salt such as aluminum hydroxidegel or aluminum phosphate or alum, but may also be a salt of calcium,magnesium, iron or zinc, or may be an insoluble suspension of acylatedtyrosine, or acylated sugars, cationically or anionically derivatizedsaccharides, or polyphosphazenes. In one embodiment, the fusion protein,PE or PilA may be adsorbed onto aluminium phosphate. In anotherembodiment, the fusion protein, PE or PilA may be adsorbed ontoaluminium hydroxide. In a third embodiment, alum may be used as anadjuvant.

Suitable adjuvant systems which promote a predominantly Th1 responseinclude: non-toxic derivatives of lipid A, Monophosphoryl lipid A (MPL)or a derivative thereof, particularly 3-de-O-acylated monophosphoryllipid A (3D-MPL) (for its preparation see GB 2220211 A); and acombination of monophosphoryl lipid A, preferably 3-de-O-acylatedmonophosphoryl lipid A, together with either an aluminum salt (forinstance aluminum phosphate or aluminum hydroxide) or an oil-in-wateremulsion. In such combinations, antigen and 3D-MPL are contained in thesame particulate structures, allowing for more efficient delivery ofantigenic and immunostimulatory signals. Studies have shown that 3D-MPLis able to further enhance the immunogenicity of an alum-adsorbedantigen (Thoelen et al. Vaccine (1998) 16:708-14; EP 689454-B1).

AS01 is an Adjuvant System containing MPL (3-O-desacyl-4′-monophosphoryllipid A), QS21 (Quillaja saponaria Molina, fraction 21) Antigenics, NewYork, N.Y., USA) and liposomes. AS01B is an Adjuvant System containingMPL, QS21 and liposomes (50 μg MPL and 50 μg QS21). AS01E is an AdjuvantSystem containing MPL, QS21 and liposomes (25 μg MPL and 25 μg QS21). Inone embodiment, the immunogenic composition or vaccine comprises AS01.In another embodiment, the immunogenic composition or vaccine comprisesAS01B or AS01E. In a particular embodiment, the immunogenic compositionor vaccine comprises AS01E.

AS03 is an Adjuvant System containing α-Tocopherol and squalene in anoil/water (o/w) emulsion. AS03_(A) is an Adjuvant System containingα-Tocopherol and squalene in an o/w emulsion (11.86 mg tocopherol).AS03_(B) is an Adjuvant System containing α-Tocopherol and squalene inan o/w emulsion (5.93 mg tocopherol). AS03c is an Adjuvant Systemcontaining α-Tocopherol and squalene in an o/w emulsion (2.97 mgtocopherol). In one embodiment, the immunogenic composition or vaccinecomprises AS03.

AS04 is an Adjuvant System containing MPL (50 μg MPL) adsorbed on analuminum salt (500 μg Al³⁺). In one embodiment, the immunogeniccomposition or vaccine comprises AS04.

A system involving the use of QS21 and 3D-MPL is disclosed in WO94/00153. A composition wherein the QS21 is quenched with cholesterol isdisclosed in WO 96/33739. An additional adjuvant formulation involvingQS21, 3D-MPL and tocopherol in an oil in water emulsion is described inWO 95/17210. In one embodiment the immunogenic composition additionallycomprises a saponin, which may be QS21. The formulation may alsocomprise an oil in water emulsion and tocopherol (WO 95/17210).Unmethylated CpG containing oligonucleotides (WO 96/02555) and otherimmunomodulatory oligonucleotides (WO 0226757 and WO 03507822) are alsopreferential inducers of a TH1 response and are suitable for use in thepresent invention.

Additional adjuvants are those selected from the group of metal salts,oil in water emulsions, Toll like receptor agonists, (in particular Tolllike receptor 2 agonist, Toll like receptor 3 agonist, Toll likereceptor 4 agonist, Toll like receptor 7 agonist, Toll like receptor 8agonist and Toll like receptor 9 agonist), saponins or combinationsthereof.

The present invention provides a process for preparing an immunogeniccomposition comprising combining a fusion protein of formula (I) with anadjuvant.

The present invention further provides a vaccine containing animmunogenic composition of the invention and a pharmaceuticallyacceptable excipient.

Possible excipients include arginine, pluronic acid and/or polysorbate.In a preferred embodiment, polysorbate 80 (for example, TWEEN® 80) isused. In a further embodiment, a final concentration of about 0.03% toabout 0.06% is used. Specifically, a final concentration of about 0.03%,0.04%, 0.05% or 0.06% polysorbate 80 (w/v) may be used.

The present invention provides a process for preparing an immunogeniccomposition or vaccine comprising combining a fusion protein of formula(I) with a pharmaceutically acceptable excipient.

The present invention also provides nucleic acids encoding the proteinsof the invention. The term “nucleic acid” refers to a polymeric form ofnucleotides. Nucleotides can be ribonucleotides, deoxyribonucleotides,or modified forms of either ribonucleotides or deoxyribonucleotides. Theterm includes single and double forms of DNA. The nucleic acids arepreferably substantially free from other nucleic acids.

The present invention provides a process of producing nucleic acids ofthe invention. Nucleic acids of the invention may be prepared by methodsknown by those skilled in the art. For example, the nucleic acids of theinvention may be synthesized in part or in whole. The nucleic acids maybe prepared by digesting longer amino acids or joining shorter aminoacids.

The following examples are intended for illustration only and are notintended to limit the scope of the invention in any way.

In the examples, the following terms have the designated meaning:

-   6×his=six histidines;-   xg=centrifugal force (number gravities)-   ATP=adenosine triphosphate;-   BCA=bicinchoninic acid;-   BSA=bovine serum albumin;-   ° C.=degrees Celsius;-   CaCl₂=calcium chloride;-   CV=column volume;-   DNA=deoxyribonucleic acid;-   DSC=differential scanning calorimetry;-   DTT=dithiothreitol;-   dNTP=deoxynucleoside triphosphate;-   EDTA=ethylenediaminetetraacetic acid;-   FT=flow through;-   HCl=hydrogen chloride;-   His=his=histidine;-   HEPES=4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid;-   IMAC=immobilized metal affinity chromatography;-   IPTG=isopropyl β-D-1-thiogalactopyranoside;-   KCl=potassium chloride;-   K₂HPO₄=dibasic potassium phosphate;-   KH₂PO₄=monobasic potassium phosphate;-   LDS=lithium dodecyl sulfate;-   L=liter;-   MES=2-(N-morpholino)ethanesulfonic acid;-   MgCl₂=magnesium chloride;-   ml=milliliter;-   RPM=revolutions per minute;-   min=minute;-   mM=millimolar;-   μL=microliter;-   NaCl=sodium chloride;-   Na₂HPO₄=dibasic sodium phosphate;-   NaH₂PO₄=monobasic sodium phosphate;-   ng=nanogram;-   nm=nanometer;-   O/N=overnight;-   PBS=phosphate buffered saline;-   PCR=polymerase chain reaction;-   SB=sample buffer;-   sec=second;-   w/v=weight/volume.

EXAMPLES Example 1: Fusion Proteins

Fusion proteins were produced with different signal peptides and aminoacid linker sequences. These fusion proteins allowed for secretion ofboth Protein E and PilA (or fragments thereof) without being restrictedto a single bacterial strain. The fusion protein is released into theperiplasm after removal of the heterologous signal peptide by a signalpeptide peptidase. Fusion protein purified from the bacteria does notcontain the heterologous signal peptide. “Purified” proteins are removedfrom the bacteria and lack the signal peptide.

The following table describes fusion protein constructs made.

TABLE 3  Fusion Protein Constructs containing PilA and Protein E.

sp = signal peptide; A.A. = amino acid

The DNA and amino acid sequences for each of the signal peptides andplasmids listed in Table 3 are set forth below.

Signal Sequences:

pelB signal peptide (DNA)-SEQ ID NO. 129:atgaaatacctgctgccgaccgctgctgctggtctgctgctcctcgctgc ccagccggcgatggccpelB signal peptide (Amino Acid)-SEQ ID NO. 130:MKYLLPTAAA GLLLLAAQPA MA FlgI signal peptide (DNA)-SEQ ID NO. 131:atgattaaatttctctctgcattaattcttctactggtcacgacggcggc tcaggctFlgI signal peptide (Amino Acid)-SEQ ID NO. 132: MIKFLSALIL LLVTTAAQANadA signal peptide (DNA)-SEQ ID NO. 133:atgaaacactttccatccaaagtactgaccacagccatccttgccacttt ctgtagcggcgcactggcaNadA signal peptide (Amino Acid)-SEQ ID NO. 134:MKHFPSKVLT TAILATFCSG ALAFusion Protein Construct Sequences:

The single underlined portion of the amino acid sequences is from PilAfrom Haemophilus influenzae strain 86-028NP. The embolded underlinedportion of the amino acid sequences was derived from Protein E fromHaemophilus influenza strain 772.

LVL312 (DNA) - SEQ ID NO. 135:atgattaaatttctctctgcattaattcttctactggtcacgacggcggctcaggctgagactaaaaaagcagcggtatctgaattactgcaagcgtcagcgccttataaggctgatgtggaattatgtgtatatagcacaaatgaaacaacaaactgtacgggtggaaaaaatggtattgcagcagatataaccacagcaaaaggctatgtaaaatcagtgacaacaagcaacggtgcaataacagtaaaaggggatggcacattggcaaatatggaatatattttgcaagctacaggtaatgctgcaacaggtgtaacttggacaacaacttgcaaaggaacggatgcctctttatttccagcaaatttttgcggaagtgtcacacaaggcggcgcgcagattcagaaggctgaacaaaatgatgtgaagctggcaccgccgactgatgtacgaagcggatatatacgtttggtaaagaatgtgaattattacatcgatagtgaatcgatctgggtggataaccaagagccacaaattgtacattttgatgcagtggtgaatttagataagggattgtatgtttatcctgagcctaaacgttatgcacgttctgttcgtcagtataagatcttgaattgtgcaaattatcatttaactcaagtacgaactgatttctatgatgaattttggggacagggtttgcgggcagcacctaaaaagcaaaagaaacatacgttaagtttaacacctgatacaacgctttataatgctgctcagattatttgtgcgaactatggtgaagcattttcagttgataaaaaaggcggccaccaccaccaccaccactaaLVL312 (protein): (flgI sp)(E)(PilA aa 40-149)(GG)(ProtE aa 18-160)(GGHHHHHH) - SEQ IDNO. 136

LVL291 (DNA) - SEQ ID NO. 137:atgaaatacctgctgccgaccgctgctgctggtctgctgctcctcgctgcccagccggcgatggcccagattcagaaggctgaacaaaatgatgtgaagctggcaccgccgactgatgtacgaagcggatatatacgtttggtaaagaatgtgaattattacatcgatagtgaatcgatctgggtggataaccaagagccacaaattgtacattttgatgcagtggtgaatttagataagggattgtatgtttatcctgagcctaaacgttatgcacgttctgttcgtcagtataagatcttgaattgtgcaaattatcatttaactcaagtacgaactgatttctatgatgaattttggggacagggtttgcgggcagcacctaaaaagcaaaagaaacatacgttaagtttaacacctgatacaacgctttataatgctgctcagattatttgtgcgaactatggtgaagcattttcagttgataaaaaaggcggcactaaaaaagcagcggtatctgaattactgcaagcgtcagcgccttataaggctgatgtggaattatgtgtatatagcacaaatgaaacaacaaactgtacgggtggaaaaaatggtattgcagcagatataaccacagcaaaaggctatgtaaaatcagtgacaacaagcaacggtgcaataacagtaaaaggggatggcacattggcaaatatggaatatattttgcaagctacaggtaatgctgcaacaggtgtaacttggacaacaacttgcaaaggaacggatgcctctttatttccagcaaatttttgcggaagtgtcacacaaggcggccaccaccaccaccaccactaaLVL291 (Protein)(pelB sp)(ProtE aa 19-160)(GG)(PilA aa40-149)(GGHHHHHH) - SEQ ID NO. 138

LVL268 (DNA) - SEQ ID NO. 139:atgaaatacctgctgccgaccgctgctgctggtctgctgctcctcgctgcccagccggcgatggccgatattcagaaggctgaacaaaatgatgtgaagctggcaccgccgactgatgtacgaagcggatatatacgtttggtaaagaatgtgaattattacatcgatagtgaatcgatctgggtggataaccaagagccacaaattgtacattttgatgcagtggtgaatttagataagggattgtatgtttatcctgagcctaaacgttatgcacgttctgttcgtcagtataagatcttgaattgtgcaaattatcatttaactcaagtacgaactgatttctatgatgaattttggggacagggtttgcgggcagcacctaaaaagcaaaagaaacatacgttaagtttaacacctgatacaacgctttataatgctgctcagattatttgtgcgaactatggtgaagcattttcagttgataaaaaaggcggcactaaaaaagcagcggtatctgaattactgcaagcgtcagcgccttataaggctgatgtggaattatgtgtatatagcacaaatgaaacaacaaactgtacgggtggaaaaaatggtattgcagcagatataaccacagcaaaaggctatgtaaaatcagtgacaacaagcaacggtgcaataacagtaaaaggggatggcacattggcaaatatggaatatattttgcaagctacaggtaatgctgcaacaggtgtaacttggacaacaacttgcaaaggaacggatgcctctttatttccagcaaatttttgcggaagtgtcacacaaggcggccaccaccaccaccaccacLVL268 (protein): (pelB sp)(D)(ProtE aa 20-160)(GG)(PilA aa40-149)(GGHHHHHH) - SEQ IDNO. 140:

LVL269 (DNA) - SEQ ID NO. 141:atgaaacactttccatccaaagtactgaccacagccatccttgccactttctgtagcggcgcactggcagccacaaacgacgacgataaggctgaacaaaatgatgtgaagctggcaccgccgactgatgtacgaagcggatatatacgtttggtaaagaatgtgaattattacatcgatagtgaatcgatctgggtggataaccaagagccacaaattgtacattttgatgcagtggtgaatttagataagggattgtatgtttatcctgagcctaaacgttatgcacgttctgttcgtcagtataagatcttgaattgtgcaaattatcatttaactcaagtacgaactgatttctatgatgaattttggggacagggtttgcgggcagcacctaaaaagcaaaagaaacatacgttaagtttaacacctgatacaacgctttataatgctgctcagattatttgtgcgaactatggtgaagcattttcagttgataaaaaaggcggcactaaaaaagcagcggtatctgaattactgcaagcgtcagcgccttataaggctgatgtggaattatgtgtatatagcacaaatgaaacaacaaactgtacgggtggaaaaaatggtattgcagcagatataaccacagcaaaaggctatgtaaaatcagtgacaacaagcaacggtgcaataacagtaaaaggggatggcacattggcaaatatggaatatattttgcaagctacaggtaatgctgcaacaggtgtaacttggacaacaacttgcaaaggaacggatgcctctttatttccagcaaatttttgcggaagtgtcacacaaggcggccaccaccaccaccaccactaaLVL269 (protein): (nadA sp)(ATNDDD)(ProtE aa 22-160)(GG)(PilA aa 40-149)(GGHHHHHH) -SEQ ID NO. 142

LVL270 (DNA) - SEQ ID NO. 143:atgcaccaccaccaccaccacagcgcgcagattcagaaggctgaacaaaatgatgtgaagctggcaccgccgactgatgtacgaagcggatatatacgtttggtaaagaatgtgaattattacatcgatagtgaatcgatctgggtggataaccaagagccacaaattgtacattttgatgcagtggtgaatttagataagggattgtatgtttatcctgagcctaaacgttatgcacgttctgttcgtcagtataagatcttgaattgtgcaaattatcatttaactcaagtacgaactgatttctatgatgaattttggggacagggtttgcgggcagcacctaaaaagcaaaagaaacatacgttaagtttaacacctgatacaacgctttataatgctgctcagattatttgtgcgaactatggtgaagcattttcagttgataaaaaaggcggcactaaaaaagcagcggtatctgaattactgcaagcgtcagcgccttataaggctgatgtggaattatgtgtatatagcacaaatgaaacaacaaactgtacgggtggaaaaaatggtattgcagcagatataaccacagcaaaaggctatgtaaaatcagtgacaacaagcaacggtgcaataacagtaaaaggggatggcacattggcaaatatggaatatattttgcaagctacaggtaatgctgcaacaggtgtaacttggacaacaacttgcaaaggaacggatgcctctttatttccagcaaatttttgcggaagtgtcacacaataaLVL270 (protein): (MHHHHHH)(ProtE aa 17-160)(GG)(PilA aa40-149) - SEQ ID NO. 144:

LVL315 (DNA) - SEQ ID NO. 145:atgaaatacctgctgccgaccgctgctgctggtctgctgctcctcgctgcccagccggcgatggccatggataaggctgaacaaaatgatgtgaagctggcaccgccgactgatgtacgaagcggatatatacgtttggtaaagaatgtgaattattacatcgatagtgaatcgatctgggtggataaccaagagccacaaattgtacattttgatgcagtggtgaatttagataagggattgtatgtttatcctgagcctaaacgttatgcacgttctgttcgtcagtataagatcttgaattgtgcaaattatcatttaactcaagtacgaactgatttctatgatgaattttggggacagggtttgcgggcagcacctaaaaagcaaaagaaacatacgttaagtttaacacctgatacaacgctttataatgctgctcagattatttgtgcgaactatggtgaagcattttcagttgataaaaaaggcggcactaaaaaagcagcggtatctgaattactgcaagcgtcagcgccttataaggctgatgtggaattatgtgtatatagcacaaatgaaacaacaaactgtacgggtggaaaaaatggtattgcagcagatataaccacagcaaaaggctatgtaaaatcagtgacaacaagcaacggtgcaataacagtaaaaggggatggcacattggcaaatatggaatatattttgcaagctacaggtaatgctgcaacaggtgtaacttggacaacaacttgcaaaggaacggatgcctctttatttccagcaaatttttgcggaagtgtcacacaaggcggccaccaccaccaccaccactaaLVL315 (protein): (pelB sp)(MD)(ProtE aa 22-160)(GG)(PilA aa40-149)(GGHHHHHH) - SEQID NO. 146:

LVL317 (DNA) - SEQ ID NO. 147:atgaaatacctgctgccgaccgctgctgctggtctgctgctcctcgctgcccagccggcgatggcccagattcagaaggctgaacaaaatgatgtgaagctggcaccgccgactgatgtacgaagcggatatatacgtttggtaaagaatgtgaattattacatcgatagtgaatcgatctgggtggataaccaagagccacaaattgtacattttgatgcagtggtgaatttagataagggattgtatgtttatcctgagcctaaacgttatgcacgttctgttcgtcagtataagatcttgaattgtgcaaattatcatttaactcaagtacgaactgatttctatgatgaattttggggacagggtttgcgggcagcacctaaaaagcaaaagaaacatacgttaagtttaacacctgatacaacgctttataatgctgctcagattatttgtgcgaactatggtgaagcattttcagttgataaaaaaggcggcactaaaaaagcagcggtatctgaattactgcaagcgtcagcgccttataaggctgatgtggaattatgtgtatatagcacaaatgaaacaacaaactgtacgggtggaaaaaatggtattgcagcagatataaccacagcaaaaggctatgtaaaatcagtgacaacaagcaacggtgcaataacagtaaaaggggatggcacattggcaaatatggaatatattttgcaagctacaggtaatgctgcaacaggtgtaacttggacaacaacttgcaaaggaacggatgcctctttatttccagcaaatttttgcggaagtgtcacacaataaLVL317 (protein): (pelB sp)(ProtE aa 19-160)(GG)(PilA aa40-149) - SEQ ID NO. 148:

LVL318 (DNA) - SEQ ID NO. 149:atgaaatacctgctgccgaccgctgctgctggtctgctgctcctcgctgcccagccggcgatggccatggataaggctgaacaaaatgatgtgaagctggcaccgccgactgatgtacgaagcggatatatacgtttggtaaagaatgtgaattattacatcgatagtgaatcgatctgggtggataaccaagagccacaaattgtacattttgatgcagtggtgaatttagataagggattgtatgtttatcctgagcctaaacgttatgcacgttctgttcgtcagtataagatcttgaattgtgcaaattatcatttaactcaagtacgaactgatttctatgatgaattttggggacagggtttgcgggcagcacctaaaaagcaaaagaaacatacgttaagtttaacacctgatacaacgctttataatgctgctcagattatttgtgcgaactatggtgaagcattttcagttgataaaaaaggcggcactaaaaaagcagcggtatctgaattactgcaagcgtcagcgccttataaggctgatgtggaattatgtgtatatagcacaaatgaaacaacaaactgtacgggtggaaaaaatggtattgcagcagatataaccacagcaaaaggctatgtaaaatcagtgacaacaagcaacggtgcaataacagtaaaaggggatggcacattggcaaatatggaatatattttgcaagctacaggtaatgctgcaacaggtgtaacttggacaacaacttgcaaaggaacggatgcctctttatttccagcaaatttttgcggaagtgtcacacaataaLVL318 (protein): (pelB sp)(MD)(ProtE aa 22-160)(GG)(PilA aa40-149) - SEQ ID NO. 150:

LVL702 (DNA) - SEQ ID NO. 181:atgaaatacctgctgccgaccgctgctgctggtctgctgctcctcgctgcccagccggcgatggccattcagaaggctgaacaaaatgatgtgaagctggcaccgccgactgatgtacgaagcggatatatacgtttggtaaagaatgtgaattattacatcgatagtgaatcgatctgggtggataaccaagagccacaaattgtacattttgatgcagtggtgaatttagataagggattgtatgtttatcctgagcctaaacgttatgcacgttctgttcgtcagtataagatcttgaattgtgcaaattatcatttaactcaagtacgaactgatttctatgatgaattttggggacagggtttgcgggcagcacctaaaaagcaaaagaaacatacgttaagtttaacacctgatacaacgctttataatgctgctcagattatttgtgcgaactatggtgaagcattttcagttgataaaaaaggcggcactaaaaaagcagcggtatctgaattactgcaagcgtcagcgccttataaggctgatgtggaattatgtgtatatagcacaaatgaaacaacaaactgtacgggtggaaaaaatggtattgcagcagatataaccacagcaaaaggctatgtaaaatcagtgacaacaagcaacggtgcaataacagtaaaaggggatggcacattggcaaatatggaatatattttgcaagctacaggtaatgctgcaacaggtgtaacttggacaacaacttgcaaaggaacggatgcctctttatttccagcaaatttttgcggaagtgtcacacaaggcggccaccaccaccaccaccacLVL702 (protein): (pelB sp)(ProtE aa 20-160)(GG)(PilA aa40-149)(GGHHHHHH) - SEQ IDNO. 182:

LVL736 (DNA) - SEQ ID NO. 183:atgaaatacctgctgccgaccgctgctgctggtctgctgctcctcgctgcccagccggcgatggccagcgcccagattcagaaggctgaacaaaatgatgtgaagctggcaccgccgactgatgtacgaagcggatatatacgtttggtaaagaatgtgaattattacatcgatagtgaatcgatctgggtggataaccaagagccacaaattgtacattttgatgcagtggtgaatttagataagggattgtatgtttatcctgagcctaaacgttatgcacgttctgttcgtcagtataagatcttgaattgtgcaaattatcatttaactcaagtacgaactgatttctatgatgaattttggggacagggtttgcgggcagcacctaaaaagcaaaagaaacatacgttaagtttaacacctgatacaacgctttataatgctgctcagattatttgtgcgaactatggtgaagcattttcagttgataaaaaaggcggcactaaaaaagcagcggtatctgaattactgcaagcgtcagcgccttataaggctgatgtggaattatgtgtatatagcacaaatgaaacaacaaactgtacgggtggaaaaaatggtattgcagcagatataaccacagcaaaaggctatgtaaaatcagtgacaacaagcaacggtgcaataacagtaaaaggggatggcacattggcaaatatggaatatattttgcaagctacaggtaatgctgcaacaggtgtaacttggacaacaacttgcaaaggaacggatgcctctttatttccagcaaatttttgcggaagtgtcacacaaggcggccaccaccaccaccaccacLVL736 (protein): (pelB sp)(ProtE aa 17-160)(GG)(PilA aa40-149)(GGHHHHHH) - SEQ IDNO. 184:

LVL737 (DNA) - SEQ ID NO. 185:atgaaatacctgctgccgaccgctgctgctggtctgctgctcctcgctgcccagccggcgatggccgcccagattcagaaggctgaacaaaatgatgtgaagctggcaccgccgactgatgtacgaagcggatatatacgtttggtaaagaatgtgaattattacatcgatagtgaatcgatctgggtggataaccaagagccacaaattgtacattttgatgcagtggtgaatttagataagggattgtatgtttatcctgagcctaaacgttatgcacgttctgttcgtcagtataagatcttgaattgtgcaaattatcatttaactcaagtacgaactgatttctatgatgaattttggggacagggtttgcgggcagcacctaaaaagcaaaagaaacatacgttaagtttaacacctgatacaacgctttataatgctgctcagattatttgtgcgaactatggtgaagcattttcagttgataaaaaaggcggcactaaaaaagcagcggtatctgaattactgcaagcgtcagcgccttataaggctgatgtggaattatgtgtatatagcacaaatgaaacaacaaactgtacgggtggaaaaaatggtattgcagcagatataaccacagcaaaaggctatgtaaaatcagtgacaacaagcaacggtgcaataacagtaaaaggggatggcacattggcaaatatggaatatattttgcaagctacaggtaatgctgcaacaggtgtaacttggacaacaacttgcaaaggaacggatgcctctttatttccagcaaatttttgcggaagtgtcacacaaggcggccaccaccaccaccaccacLVL737 (protein): (pelB sp)(ProtE aa 18-160)(GG)(PilA aa40-149)(GGHHHHHH) - SEQ IDNO. 186:

LVL738 (DNA) - SEQ ID NO. 187:atgaaatacctgctgccgaccgctgctgctggtctgctgctcctcgctgcccagccggcgatggccaaggctgaacaaaatgatgtgaagctggcaccgccgactgatgtacgaagcggatatatacgtttggtaaagaatgtgaattattacatcgatagtgaatcgatctgggtggataaccaagagccacaaattgtacattttgatgcagtggtgaatttagataagggattgtatgtttatcctgagcctaaacgttatgcacgttctgttcgtcagtataagatcttgaattgtgcaaattatcatttaactcaagtacgaactgatttctatgatgaattttggggacagggtttgcgggcagcacctaaaaagcaaaagaaacatacgttaagtttaacacctgatacaacgctttataatgctgctcagattatttgtgcgaactatggtgaagcattttcagttgataaaaaaggcggcactaaaaaagcagcggtatctgaattactgcaagcgtcagcgccttataaggctgatgtggaattatgtgtatatagcacaaatgaaacaacaaactgtacgggtggaaaaaatggtattgcagcagatataaccacagcaaaaggctatgtaaaatcagtgacaacaagcaacggtgcaataacagtaaaaggggatggcacattggcaaatatggaatatattttgcaagctacaggtaatgctgcaacaggtgtaacttggacaacaacttgcaaaggaacggatgcctctttatttccagcaaatttttgcggaagtgtcacacaaggcggccaccaccaccaccaccacLVL738 (protein): (pelB sp)(ProtE aa 22-160)(GG)(PilA aa40-149)(GGHHHHHH) - SEQ IDNO. 188:

LVL739 (DNA) - SEQ ID NO. 189:atgaaatacctgctgccgaccgctgctgctggtctgctgctcctcgctgcccagccggcgatggccgctgaacaaaatgatgtgaagctggcaccgccgactgatgtacgaagcggatatatacgtttggtaaagaatgtgaattattacatcgatagtgaatcgatctgggtggataaccaagagccacaaattgtacattttgatgcagtggtgaatttagataagggattgtatgthatcctgagcctaaacgttatgcacgttctgttcgtcagtataagatcttgaattgtgcaaattatcatttaactcaagtacgaactgatttctatgatgaattttggggacagggtttgcgggcagcacctaaaaagcaaaagaaacatacgttaagtttaacacctgatacaacgctttataatgctgctcagattatttgtgcgaactatggtgaagcattttcagttgataaaaaaggcggcactaaaaaagcagcggtatctgaattactgcaagcgtcagcgccttataaggctgatgtggaattatgtgtatatagcacaaatgaaacaacaaactgtacgggtggaaaaaatggtattgcagcagatataaccacagcaaaaggctatgtaaaatcagtgacaacaagcaacggtgcaataacagtaaaaggggatggcacattggcaaatatggaatatattttgcaagctacaggtaatgctgcaacaggtgtaacttggacaacaacttgcaaaggaacggatgcctctttatttccagcaaatttttgcggaagtgtcacacaaggcggccaccaccaccaccaccacLVL739 (protein): (pelB sp)(ProtE aa 23-160)(GG)(PilA aa40-149)(GGHHHHHH) - SEQ IDNO. 190:

LVL740 (DNA) - SEQ ID NO. 191:atgaaatacctgctgccgaccgctgctgctggtctgctgctcctcgctgcccagccggcgatggccgaacaaaatgatgtgaagctggcaccgccgactgatgtacgaagcggatatatacgtttggtaaagaatgtgaattattacatcgatagtgaatcgatctgggtggataaccaagagccacaaattgtacattttgatgcagtggtgaatttagataagggattgtatgtttatcctgagcctaaacgttatgcacgttctgttcgtcagtataagatcttgaattgtgcaaattatcatttaactcaagtacgaactgatttctatgatgaattttggggacagggtttgcgggcagcacctaaaaagcaaaagaaacatacgttaagtttaacacctgatacaacgctttataatgctgctcagattatttgtgcgaactatggtgaagcattttcagttgataaaaaaggcggcactaaaaaagcagcggtatctgaattactgcaagcgtcagcgccttataaggctgatgtggaattatgtgtatatagcacaaatgaaacaacaaactgtacgggtggaaaaaatggtattgcagcagatataaccacagcaaaaggctatgtaaaatcagtgacaacaagcaacggtgcaataacagtaaaaggggatggcacattggcaaatatggaatatattttgcaagctacaggtaatgctgcaacaggtgtaacttggacaacaacttgcaaaggaacggatgcctctttatttccagcaaatttttgcggaagtgtcacacaaggcggccaccaccaccaccaccacLVL740 (protein): (pelB sp)(ProtE aa 24-160)(GG)(PilA aa40-149)(GGHHHHHH) - SEQ IDNO. 192:

LVL735 (DNA) - SEQ ID NO. 193:atgaaatacctgctgccgaccgctgctgctggtctgctgctcctcgctgcccagccggcgatggccattcagaaggctgaacaaaatgatgtgaagctggcaccgccgactgatgtacgaagcggatatatacgtttggtaaagaatgtgaattattacatcgatagtgaatcgatctgggtggataaccaagagccacaaattgtacattttgatgcagtggtgaatttagataagggattgtatgtttatcctgagcctaaacgttatgcacgttctgttcgtcagtataagatcttgaattgtgcaaattatcatttaactcaagtacgaactgatttctatgatgaattttggggacagggtttgcgggcagcacctaaaaagcaaaagaaacatacgttaagtttaacacctgatacaacgctttataatgctgctcagattatttgtgcgaactatggtgaagcattttcagttgataaaaaaggcggcactaaaaaagcagcggtatctgaattactgcaagcgtcagcgccttataaggctgatgtggaattatgtgtatatagcacaaatgaaacaacaaactgtacgggtggaaaaaatggtattgcagcagatataaccacagcaaaaggctatgtaaaatcagtgacaacaagcaacggtgcaataacagtaaaaggggatggcacattggcaaatatggaatatattttgcaagctacaggtaatgctgcaacaggtgtaacttggacaacaacttgcaaaggaacggatgcctctttatttccagcaaatttttgcggaagtgtcacacaaLVL735 (protein): (pelB sp)(ProtE aa 20-160)(GG)(PilA aa40-149) - SEQ ID NO. 194:

LVL778 (DNA) - SEQ ID NO. 195:atgaaatacctgctgccgaccgctgctgctggtctgctgctcctcgctgcccagccggcgatggccagcgcccagattcagaaggctgaacaaaatgatgtgaagctggcaccgccgactgatgtacgaagcggatatatacgtttggtaaagaatgtgaattattacatcgatagtgaatcgatctgggtggataaccaagagccacaaattgtacattttgatgcagtggtgaatttagataagggattgtatgtttatcctgagcctaaacgttatgcacgttctgttcgtcagtataagatcttgaattgtgcaaattatcatttaactcaagtacgaactgatttctatgatgaattttggggacagggtttgcgggcagcacctaaaaagcaaaagaaacatacgttaagtttaacacctgatacaacgctttataatgctgctcagattatttgtgcgaactatggtgaagcattttcagttgataaaaaaggcggcactaaaaaagcagcggtatctgaattactgcaagcgtcagcgccttataaggctgatgtggaattatgtgtatatagcacaaatgaaacaacaaactgtacgggtggaaaaaatggtattgcagcagatataaccacagcaaaaggctatgtaaaatcagtgacaacaagcaacggtgcaataacagtaaaaggggatggcacattggcaaatatggaatatattttgcaagctacaggtaatgctgcaacaggtgtaacttggacaacaacttgcaaaggaacggatgcctctttatttccagcaaatttttgcggaagtgtcacacaaLVL778 (protein): (pelB sp)(ProtE aa 17-160)(GG)(PilA aa40-149) - SEQ ID NO. 196:

LVL779 (DNA) - SEQ ID NO. 197:atgaaatacctgctgccgaccgctgctgctggtctgctgctcctcgctgcccagccggcgatggccgcccagattcagaaggctgaacaaaatgatgtgaagctggcaccgccgactgatgtacgaagcggatatatacgtttggtaaagaatgtgaattattacatcgatagtgaatcgatctgggtggataaccaagagccacaaattgtacattttgatgcagtggtgaatttagataagggattgtatgtttatcctgagcctaaacgttatgcacgttctgttcgtcagtataagatcttgaattgtgcaaattatcatttaactcaagtacgaactgatttctatgatgaattttggggacagggtttgcgggcagcacctaaaaagcaaaagaaacatacgttaagtttaacacctgatacaacgctttataatgctgctcagattatttgtgcgaactatggtgaagcattttcagttgataaaaaaggcggcactaaaaaagcagcggtatctgaattactgcaagcgtcagcgccttataaggctgatgtggaattatgtgtatatagcacaaatgaaacaacaaactgtacgggtggaaaaaatggtattgcagcagatataaccacagcaaaaggctatgtaaaatcagtgacaacaagcaacggtgcaataacagtaaaaggggatggcacattggcaaatatggaatatattttgcaagctacaggtaatgctgcaacaggtgtaacttggacaacaacttgcaaaggaacggatgcctctttatttccagcaaatttttgcggaagtgtcacacaaLVL779 (protein): (pelB sp)(ProtE aa 18-160)(GG)(PilA aa40-149) - SEQ ID NO. 198:

LVL780 (DNA) - SEQ ID NO. 199:atgaaatacctgctgccgaccgctgctgctggtctgctgctcctcgctgcccagccggcgatggccaaggctgaacaaaatgatgtgaagctggcaccgccgactgatgtacgaagcggatatatacgtttggtaaagaatgtgaattattacatcgatagtgaatcgatctgggtggataaccaagagccacaaattgtacattttgatgcagtggtgaatttagataagggattgtatgtttatcctgagcctaaacgttatgcacgttctgttcgtcagtataagatcttgaattgtgcaaattatcatttaactcaagtacgaactgatttctatgatgaattttggggacagggtttgcgggcagcacctaaaaagcaaaagaaacatacgttaagtttaacacctgatacaacgctttataatgctgctcagattatttgtgcgaactatggtgaagcattttcagttgataaaaaaggcggcactaaaaaagcagcggtatctgaattactgcaagcgtcagcgccttataaggctgatgtggaattatgtgtatatagcacaaatgaaacaacaaactgtacgggtggaaaaaatggtattgcagcagatataaccacagcaaaaggctatgtaaaatcagtgacaacaagcaacggtgcaataacagtaaaaggggatggcacattggcaaatatggaatatattttgcaagctacaggtaatgctgcaacaggtgtaacttggacaacaacttgcaaaggaacggatgcctctttatttccagcaaatttttgcggaagtgtcacacaaLVL780 (protein): (pelB sp)(ProtE aa 22-160)(GG)(PilA aa40-149) - SEQ ID NO. 200:

LVL781 (DNA) - SEQ ID NO. 201:atgaaatacctgctgccgaccgctgctgctggtctgctgctcctcgctgcccagccggcgatggccgctgaacaaaatgatgtgaagctggcaccgccgactgatgtacgaagcggatatatacgtttggtaaagaatgtgaattattacatcgatagtgaatcgatctgggtggataaccaagagccacaaattgtacattttgatgcagtggtgaatttagataagggattgtatgthatcctgagcctaaacgttatgcacgttctgttcgtcagtataagatcttgaattgtgcaaattatcatttaactcaagtacgaactgatttctatgatgaattttggggacagggtttgcgggcagcacctaaaaagcaaaagaaacatacgttaagtttaacacctgatacaacgctttataatgctgctcagattatttgtgcgaactatggtgaagcattttcagttgataaaaaaggcggcactaaaaaagcagcggtatctgaattactgcaagcgtcagcgccttataaggctgatgtggaattatgtgtatatagcacaaatgaaacaacaaactgtacgggtggaaaaaatggtattgcagcagatataaccacagcaaaaggctatgtaaaatcagtgacaacaagcaacggtgcaataacagtaaaaggggatggcacattggcaaatatggaatatattttgcaagctacaggtaatgctgcaacaggtgtaacttggacaacaacttgcaaaggaacggatgcctctttatttccagcaaatttttgcggaagtgtcacacaaLVL781 (protein): (pelB sp)(ProtE aa 23-160)(GG)(PilA aa40-149) - SEQ ID NO. 202:

LVL782 (DNA) - SEQ ID NO. 203:atgaaatacctgctgccgaccgctgctgctggtctgctgctcctcgctgcccagccggcgatggccgaacaaaatgatgtgaagctggcaccgccgactgatgtacgaagcggatatatacgtttggtaaagaatgtgaattattacatcgatagtgaatcgatctgggtggataaccaagagccacaaattgtacattttgatgcagtggtgaatttagataagggattgtatgtttatcctgagcctaaacgttatgcacgttctgttcgtcagtataagatcttgaattgtgcaaattatcatttaactcaagtacgaactgatttctatgatgaattttggggacagggtttgcgggcagcacctaaaaagcaaaagaaacatacgttaagtttaacacctgatacaacgctttataatgctgctcagattatttgtgcgaactatggtgaagcattttcagttgataaaaaaggcggcactaaaaaagcagcggtatctgaattactgcaagcgtcagcgccttataaggctgatgtggaattatgtgtatatagcacaaatgaaacaacaaactgtacgggtggaaaaaatggtattgcagcagatataaccacagcaaaaggctatgtaaaatcagtgacaacaagcaacggtgcaataacagtaaaaggggatggcacattggcaaatatggaatatattttgcaagctacaggtaatgctgcaacaggtgtaacttggacaacaacttgcaaaggaacggatgcctctttatttccagcaaatttttgcggaagtgtcacacaaLVL782 (protein): (pelB sp)(ProtE aa 24-160)(GG)(PilA aa40-149) - SEQ ID NO. 204:

The full length sequence for PE and PilA from which the above sequenceswere obtained are set forth in SEQ ID NO. 4 (PE) and SEQ ID NO. 58(PilA), respectively.

Example 2: Vector Construction and Transformation

Primers for amplifying PE from H. influenzae strain 772 were designedbased on the sequence of H. influenzae strain Hi Rd. The 5′ primersequence contains one nucleotide difference compared to the NTHi 772sequence, introducing an amino acid difference at position 24 whencompared with the currently reported NTHi 772 genome sequence. Aminoacid #24 in the fusion protein constructs is E (glutamic acid) insteadof K (lysine) as found in NTHi 772.

DNA Sequence for PE from H. influenzae strain Rd. SEQ ID NO. 151atgaaaaaaattattttaacattatcacttgggttacttaccgcttgttctgctcaaatccaaaaggctgaacaaaatgatgtgaagctggcaccgccgactgatgtacgaagcggatatatacgtttggtaaagaatgtgaattattacatcgatagtgaatcgatctgggtggataaccaagagccacaaattgtacattttgatgctgtggtgaatttagataggggattgtatgtttatcctgagcctaaacgttatgcacgttctgttcgtcagtataagattttgaattgtgcaaattatcatttaactcaaatacgaactgatttctatgatgaattttggggacagggtttgcgggcagcacctaaaaagcaaaagaaacatacgttaagtttaacacctgatacaacgctttataatgctgctcagattatttgtgcaaattatggtaaagcattttcagttgataaaaaataaProtein Sequence for PE from H. influenzae strain Rd. SEQ ID NO. 152MKKIILTLSL GLLTACSAQI QKAEQNDVKL APPTDVRSGYIRLVKNVNYY IDSESIWVDN QEPQIVHFDA VVNLDRGLYVYPEPKRYARS VRQYKILNCA NYHLTQIRTD FYDEFWGQGLRAAPKKQKKH TLSLTPDTTL YNAAQIICAN YGKAFSVDKKDNA Sequence for PE from H. influenzae strain 772(as set forth in: Microbes & Infection,Corrigendum to “Identification of a novelHaemophilus influenzae protein important foradhesion to epithelia cells” [Microbes Infect. 10(2008) 87-97], available online Jul. 6, 2010, “Article in Press”))SEQ ID NO. 153 atgaaaaaaattattttaacattatcacttgggttacttactgcctgttctgctcaaatccaaaaggctaaacaaaatgatgtgaagctggcaccgccgactgatgtacgaagcggatatatacgtttggtaaagaatgtgaattattacatcgatagtgaatcgatctgggtggataaccaagagccacaaattgtacattttgatgcagtggtgaatttagataagggattgtatgtttatcctgagcctaaacgttatgcacgttctgttcgtcagtataagatcttgaattgtgcaaattatcatttaactcaagtacgaactgatttctatgatgaattttggggacagggtttgcgggcagcacctaaaaagcaaaagaaacatacgttaagtttaacacctgatacaacgctttataatgctgctcagattatttgtgcgaactatggtgaagcattttcagttgataaaaaaProtein Sequence for PE from H. influenzae strain772 (as set forth in: Microbes & Infection,Corrigendum to “Identification of a novelHaemophilus influenzae protein important foradhesion to epithelia cells” [Microbes Infect. 10(2008) 87-97], available online Jul. 6, 2010, “Article in Press”))SEQ ID NO. 154 MKKIILTLSL GLLTACSAQI QKAKQNDVKL APPTDVRSGYIRLVKNVNYY IDSESIWVDN QEPQIVHFDA VVNLDKGLYVYPEPKRYARS VRQYKILNCA NYHLTQVRTD FYDEFWGQGLRAAPKKQKKH TLSLTPDTTL YNAAQIICAN YGEAFSVDKKVector Construction:

To generate LVL312, LVL291, LVL268, LVL269, LVL270, LVL702, LVL735,LVL778, LVL779, LVL780, LVL781 and LVL782, a polymerase chain reaction(PCR) preparation of the following components was prepared (specificcomponents are subsequently exemplified): 36.6 μl of deionized water, 5μl of buffer #1 10×, 5 μl of dNTPs 2 mM, 2 μl MgCl₂ 25 mM, 0.4 μl ofprimer #1 (50 μM), 0.4 μl of primer #2 (50 μM), 0.5 μl of template (100ng/μl) and 0.4 μl of KOD HiFi DNA polymerase 2.5 units/μl (NOVAGEN®) wasformulated. Polymerase chain reaction involved 25 cycles of 15 secondsof denaturation at 98° C., 2 seconds for annealing at 55° C. and 20seconds of primer extension at 72° C. The PCR products were purifiedusing QIAQUICK® PCR purification kit (QIAGEN®). This product was usedunder conditions recommended by the supplier which were: the addition of5 volumes Buffer PB, provided in the QIAQUICK® PCR purification kit, to1 volume of the PCR preparation. The PCR preparation with Buffer PB wassubsequently mixed by vortex. A QIAQUICK® column was placed into a 2 mlcollection tube. To bind DNA in the PCR preparation to the column, themixed sample was applied to the QIAQUICK® column and centrifuged for30-60 seconds at 14 000 RPM. The flow-through was discarded and theQIAQUICK® column was placed back in the same tube. To wash the bound DNA0.75 ml Buffer PE, provided in the QIAQUICK PCR purification kit, wasadded to the QIAQUICK® column, and the column was centrifuged for 30-60seconds at 14 000 RPM. The flow-through was discarded and the QIAQUICK®column was placed back in the same tube. The QIAQUICK® column wascentrifuged once more in the 2 ml collection tube for 1 minute to removeresidual wash buffer. Each QIAQUICK® column was placed in a clean 1.5 mlmicrocentrifuge tube. To elute the DNA, 33 μl water was added to thecenter of the QIAQUICK® membrane and the column was centrifuged for 1minute at 14 000 RPM. Restriction enzymes and buffer related wereobtained from New England BioLabs. For example, approximately 5 μl ofpET26b vector (100 ng/μl), 2 μl of NEBuffer 2 (New England Biolabs,1×NEBuffer 2: 50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl₂, 1 mMdithiothreitol, pH 7.9 at 25° C.), 1 μl of NdeI (20 000 units/ml), 1 μlof HindIII (20 000 units/ml) and 11 μl of deionized water were mixed andincubated for two hours at 37° C. for DNA digestion. Thereafter, asecond step of purification was performed using the QIAQUICK® PCRpurification kit (QIAGEN®) with the procedure described above.

Ligation was performed using Quick T4 DNA ligase and Quick LigationReaction Buffer from New England BioLabs. For example, around 10 ng ofvector and 30 ng of insert in 10 μl of deionized water were mixed with10 μl of 2× Quick Ligation Reaction Buffer (New England Biolabs, 132 mMTris-HCl, 20 mM MgCl₂, 2 mM dithiothreitol, 2 mM ATP, 15% polyethyleneglycol, pH 7.6 at 25° C.) and 1 μl of Quick T4 DNA ligase (New EnglandBiolabs). The enzymatic reaction was incubated for 5 minutes at roomtemperature before transformation.

To generate LVL315, LVL317, LVL318, LVL736, LVL737, LVL738, LVL739 andLVL740, a PCR preparation of the following components was prepared: 40μl of deionized water, 5 μl of reaction buffer 10×, 1 μl of dNTPs mix, 1μl of primer #1 (10 μM), 1 μl of primer #2 (10 μM), 1 μl of template (25ng/μl) and 1 μl of PfuUltra High-Fidelity DNA polymerase 2.5 units/μl(QuikChange II Site-Directed Mutagenesis Kit, Agilent Technologies,Stratagene Division) was formulated. Polymerase chain reaction involvedone cycle of denaturation at 95° C. for 30 sec, 18 cycles of 30 sec ofdenaturation at 95° C., 1 min for annealing at 55° C. and 5 min 30 secof primer extension at 68° C. The PCR products were digested using 1 μlof DpnI restriction enzyme at 37° C. for one hour before transformation.

A detailed list of PCR primer sequences used for amplifications isillustrated in Table 4.

To generate pRIT16711, the PE gene fragment coding for amino acids 22 to160 of SEQ ID NO. 4, which excludes the sequence coding for itscorresponding secretion signal, was amplified by PCR from genomic DNA ofNTHi strain 772. The amplification primers were designed based on theavailable strain Hi Rd sequence (at that time, the 772 sequence was notknown). The 5′ primer sequence contains one mutation compared to theNTHi 772 sequence (sequence as now available), introducing one aminoacid difference in PE coding sequence at position 24, glutamic acid (E)instead of lysine (K). After PCR amplification, the insert was cloned inthe pET-26(+) expression vector (NOVAGEN®) using BamHI and XhoIrestriction sites.

To generate pRIT16671, a DNA fragment coding for a PilA gene fragment(amino acids 40 to 149 of SEQ ID NO. 58, SEQ ID NO. 127), which excludesits leader peptide as well as a portion of the predicted hydrophobicalpha helix, was amplified from genomic DNA of NTHi strain 86-028NP andcloned into the pET15 expression vector. The vector pRIT16790(containing amino acids 40 to 149 from NTHi strain 86-028NP) was used asa template to generate the vector pRIT16671. The PilA gene fragment wasamplified by PCR using the vector pRIT16790 and primers MDES PILA-3 andMDES PILA-4. The PilA fragment was cloned into the pET-26 expressionvector using NdeI/XhoI restriction sites. The DNA sequence encoding sixhistidine (his) amino acids was incorporated into the 5′ primer to addsix histidines (6×his) at the N-terminal end of the PilA sequence (MDESPILA-3).

To generate LVL312 (FlgI signal peptide-E-PilA fragment-GG-PEfragment-GGHHHHHH), a polymerase chain reaction was performed to amplifythe PilA gene (amino acids 40-149/strain 86-028NP) using the pRIT16671vector as a template and primers CAN534 and CAN537. DNA sequencecorresponding to FlgI signal peptide (sp) and glutamic acid (E) aminoacid was incorporated into the 5′ primer (CAN534). To link the PilAsequence to PE sequence, DNA sequence corresponding to the two glycine(GG) amino acids linker and the N-terminal PE amino acids wereincorporated into the 3′ primer (CAN537). Another polymerase chainreaction was performed to amplify the PE gene (amino acids 18-160) usingpRIT16711 vector as a template and primers CAN536 and CAN538. DNAsequence corresponding to the C-terminal PilA amino acids and GG aminoacids were incorporated into the 5′ primer to link pilA to PE sequence(CAN536). DNA sequence corresponding to the GG amino acids linker and6×his amino acids were incorporated into the 3′ primer (CAN538).Finally, to generate LVL312, a third polymerase chain reaction wasperformed to amplify the PilA and PE genes in fusion with the FlgIsignal peptide at the N-terminus, a glutamic acid (E) amino acid betweenFlgI and pilA, a GG linker between PilA and PE sequences and a GG linkerbetween PE and the 6×his amino acids at the C-terminus. To achieve thisamplification, the products of the two polymerase chain reactionsdescribed above were used as a template with primers CAN534 and CAN538.DNA sequence corresponding to NdeI restriction site was incorporatedinto the 5′ primer and HindIII restriction site was incorporated intothe 3′ primer. The generated PCR product was then inserted into thepET-26b(+) cloning vector (NOVAGEN®).

To generate LVL291 (pelB signal peptide-PE fragment-GG-PilAfragment-GG-6×his), a polymerase chain reaction was performed to amplifythe PE gene (amino acids 19-160) using the pRIT16711 vector as atemplate and primers CAN544 and CAN546. DNA sequence corresponding topelB signal peptide (sp) amino acids was incorporated into the 5′ primer(CAN544). To link the PilA sequence to the PE sequence, DNA sequencecorresponding to GG amino acids linker and the N-terminal PilA aminoacids were incorporated into the 3′ primer (CAN546). Another polymerasechain reaction was performed to amplify the PilA gene (amino acids40-149 of SEQ ID NO. 58, SEQ ID NO. 127) using the pRIT16671 vector as atemplate with primers CAN545 and CAN535. DNA sequence corresponding tothe C-terminal PE amino acids and GG amino acids were incorporated intothe 5′ primer (CAN545) to link the PilA sequence to the PE sequence. DNAsequence corresponding to linker GG amino acids and 6×his amino acidswere incorporated into the 3′ primer (CAN535). Finally, to generateLVL291, a third polymerase chain reaction was performed to amplify thePE and PilA genes in fusion with the pelB signal peptide at theN-terminus, a GG linker between the PE and PilA sequences and a GGlinker between PilA and 6×his amino acids at the C-terminus. To achievethis amplification, the products of two polymerase chain reactionsdescribed above were used as a template with primers CAN544 and CAN535.DNA sequence corresponding to NdeI restriction site was incorporatedinto the 5′ primer and HindIII restriction site was incorporated intothe 3′ primer. The generated PCR product was then inserted into thepET-26b(+) cloning vector (NOVAGEN®).

To generate LVL268 (pelB signal peptide-D-PE fragment-GG-PilAfragment-GG-6×his), a polymerase chain reaction was performed to amplifythe PE gene (amino acids 20-160) using the pRIT16711 vector as atemplate with primers CAN547 and CAN546. DNA sequence corresponding tothe pelB signal peptide (sp) amino acids and aspartic acid (D) aminoacid were incorporated into the 5′ primer (CAN547). To link the PilAsequence to the PE sequence, DNA sequence corresponding to GG aminoacids linker and the N-terminal PilA amino acids were incorporated intothe 3′ primer (CAN546). Another polymerase chain reaction was performedto amplify the PilA gene (amino acids 40-149/NTHi strain 86-028NP) usingthe pRIT16671 vector as a template with CAN545 and CAN535. DNA sequencecorresponding to the C-terminal PE amino acids and GG amino acids wereincorporated into the 5′ primer (CAN545) to link the PilA sequence tothe PE sequence. DNA sequence corresponding to linker GG amino acids and6×his amino acids were incorporated into the 3′ primer (CAN535).Finally, to generate LVL268, a third polymerase chain reaction wasperformed to amplify the PE and PilA genes in fusion with the pelBsignal peptide at the N-terminus, a D amino acid between pelB signalpeptide and PE, a GG linker between PE and pilA sequences and a GGlinker between PilA and 6×his amino acids in C-term. To achieve thisamplification, the products of the two polymerase chain reactionsdescribed above were used as a template with primers CAN547 and CAN535.DNA sequence corresponding to NdeI restriction site was incorporatedinto the 5′ primer and HindIII restriction site was incorporated intothe 3′ primer. The generated PCR product was then inserted into thepET-26b(+) cloning vector (NOVAGEN®).

To generate LVL269 (NadA signal peptide-ATNDDD-PE fragment-GG-PilAfragment-GG-6×his), a polymerase chain reaction was performed to amplifythe PE gene (amino acids 22-160 of SEQ ID NO. 4) using the pRIT16711vector as a template with primers CAN548 and CAN546. DNA sequencecorresponding to pelB signal peptide (sp) amino acids and ATNDDD aminoacids were incorporated into the 5′ primer (CAN548). To link the PilAsequence to the PE sequence, DNA sequence corresponding to the GG aminoacids linker and the N-terminal PilA amino acids were incorporated intothe 3′ primer (CAN546). Another polymerase chain reaction was performedto amplify the PilA gene (amino acids 40-149 of SEQ ID NO. 58, SEQ IDNO. 127) using the pRIT16671 vector as a template with primers CAN545and CAN535. DNA sequence corresponding to the C-terminal PE amino acidsand GG amino acids were incorporated into the 5′ primer to link the PilAsequence to the PE sequence (CAN545). DNA sequence corresponding tolinker GG amino acids and 6×his amino acids were incorporated into the3′ primer (CAN535). Finally, to generate LVL269, a third polymerasechain reaction was performed to amplify the PE and PilA gene in fusionwith the NadA signal peptide at the N-terminus, ATNDDD amino acidsbetween the pelB signal peptide and PE, a GG linker between the PE andpilA sequences and a GG linker between PilA and 6×his amino acids at theC-terminus. To achieve this amplification, the products of the twopolymerase chain reactions describe above were used as a template withprimers CAN548 and CAN535. DNA sequence corresponding to NdeIrestriction site was incorporated into the 5′ primer and HindIIIrestriction site was incorporated into the 3′ primer. The generated PCRproduct was then inserted into the pET-26b(+) cloning vector (NOVAGEN®).

To generate LVL270 (M-6×His-PE fragment-GG-PilA fragment), a polymerasechain reaction was performed to amplify the PE gene (amino acids 17-160)using the pRIT16711 vector as a template with primers CAN540 and CAN542.DNA sequence corresponding to 6×his amino acids were incorporated intothe 5′ primer (CAN540). To link the PilA sequence to the PE sequence,DNA sequence corresponding to the GG amino acids linker and theN-terminal PilA amino acids were incorporated into the 3′ primer(CAN542). Another polymerase chain reaction was performed to amplify thePilA gene (amino acids 40-149/NTHi strain 86-028NP) using pRIT16671vector as a template with primers CAN541 and CAN543. DNA sequencecorresponding to the C-terminal PE amino acids and GG amino acids wereincorporated into the 5′ primer (CAN541) to link the PilA to the PEsequence. Finally, to generate LVL270, a third polymerase chain reactionwas performed to amplify the 6-his-PE-GG-PilA gene in fusion. To achievethis amplification, the products of the two polymerase chain reactionsdescribe above were used as a template with primers CAN540 and CAN543.DNA sequence corresponding to NdeI restriction site was incorporatedinto the 5′ primer and HindIII restriction site was incorporated intothe 3′ primer. The generated PCR product was then inserted into thepET-26b(+) cloning vector (NOVAGEN®).

To generate LVL315 (pelB signal peptide-MD-PE fragment-GG-PilAfragment-GG-6×his), a site-directed mutagenesis was performed to changethe N-terminal PE amino acid sequence from QIQ to MD using LVL291 as atemplate with primers CAN670 and CAN671 and the QuikChange IISite-Directed Mutagenesis Kit (Agilent Technologies, StratageneDivision).

To generate LVL317 (pelB signal peptide-PE fragment-GG-pilA fragment), asite-directed mutagenesis was performed to incorporate a stop codonbetween the PilA gene and the DNA sequence corresponding to GGHHHHHHamino acid residues (SEQ ID NO: 3) using LVL291 as a template withprimers CAN678 and CAN679 and the QuikChange II Site-DirectedMutagenesis Kit (Agilent Technologies, Stratagene Division).

To generate LVL318 (pelB signal peptide-MD-PE-GG-PilA), a site-directedmutagenesis was performed to incorporate a stop codon between the PilAgene and the DNA sequence corresponding to GGHHHHHH amino acid residues(SEQ ID NO: 3) using LVL315 as a template with primers CAN678 and CAN679and the QuikChange II Site-Directed Mutagenesis Kit (AgilentTechnologies, Stratagene Division).

To generate LVL702 (LVL291 ΔQ), a polymerase chain reaction wasperformed using the LVL291 vector as template and primers CAN1517 andCAN1518. Deletion of three nucleotides corresponding to the amino acid Qat the position 23 on LVL291 sequence was incorporated to the 5′ primer.The only difference between LVL702 and LVL291 is the deletion of aminoacid Q at the position 23 on LVL291 sequence. NdeI and HindIIIrestriction sites were incorporated into the 5′ and 3′ primersrespectively. The generated PCR product was then inserted into thepET-26b(+) cloning vector (NOVAGEN®).

To generate LVL735 (LVL317 ΔQ), a polymerase chain reaction wasperformed using the LVL317 vector as template and primers CAN1517 andCAN1519. Deletion of three nucleotides corresponding to the amino acid Qat the position 23 on LVL317 sequence was incorporated to the 5′ primer.The only difference between LVL735 and LVL317 is the deletion of aminoacid Q at the position 23 on LVL317 sequence. NdeI and HindIIIrestriction sites were incorporated into the 5′ and 3′ primersrespectively. The generated PCR product was then inserted into thepET-26b(+) cloning vector (NOVAGEN®).

To generate LVL736 (LVL291+SA), a site-directed mutagenesis wasperformed to add amino acids S and A between amino acid 22 and 23 onLVL291 sequence. LVL291 was used as template with primers CAN1531 andCAN1532 and the QuikChange II Site-Directed Mutagenesis Kit (AgilentTechnologies, Stratagene Division).

To generate LVL737 (LVL291+A), a site-directed mutagenesis was performedto add amino acid A between amino acid 22 and 23 on LVL291 sequence.LVL291 was used as template with primers CAN1529 and CAN1530 and theQuikChange II Site-Directed Mutagenesis Kit (Agilent Technologies,Stratagene Division).

To generate LVL738 (LVL291 ΔQIQ), a site-directed mutagenesis wasperformed to delete amino acids Q, I and Q at positions 23 to 25 onLVL291 sequence. LVL291 was used as template with primers CAN1523 andCAN1524 and the QuikChange II Site-Directed Mutagenesis Kit (AgilentTechnologies, Stratagene Division).

To generate LVL739 (LVL291 ΔQIQK), a site-directed mutagenesis wasperformed to delete amino acids Q, I, Q and K at positions 23 to 26 onLVL291 sequence. LVL291 was used as template with primers CAN1525 andCAN1526 and the QuikChange II Site-Directed Mutagenesis Kit (AgilentTechnologies, Stratagene Division).

To generate LVL740 (LVL291 ΔQIQKA), a site-directed mutagenesis wasperformed to delete amino acids Q, I, Q, K and A at positions 23 to 27on LVL291 sequence. LVL291 was used as template with primers CAN1527 andCAN1528 and the QuikChange II Site-Directed Mutagenesis Kit (AgilentTechnologies, Stratagene Division).

To generate LVL778 (LVL736 Δ6×His tag), LVL779 (LVL737 Δ6×His tag),LVL780 (LVL738 Δ6×His tag), LVL781 (LVL739 Δ6×His tag) and LVL782(LVL740 Δ6×His tag) a polymerase chain reaction was performed using theLVL736, LVL737, LVL738, LVL739 and LVL740 vectors as template,respectively, with primers CAN1669 and CAN543. Deletion of 6×His tagcorresponds to the amino acid sequence GGHHHHHH (SEQ ID NO. 3) at theC-terminal sequences. This deletion was incorporated to the 3′ primer.NdeI and HindIII restriction sites were incorporated into the 5′ and 3′primers respectively. The generated PCR product was then inserted intothe pET-26b(+) cloning vector (NOVAGEN®).

TABLE 4 PCR primer sequences used for PE,PilA and PE-PilA amplifications Primer ID DNA Sequence 5′-3′ CAN534CACACACATATGATTAAATTTCTCTCTGCATTAATTCTTCTACTGGTCACGACGGCGGCTCAGGCTGAGACTAAAAAA GCAGCGGTATCTG (SEQ ID NO. 155)CAN535 TGTGTGAAGCTTTTAGTGGTGGTGGTGGTGGTGGCCGCCTTGTGTGACACTTCCGCAAAAATTTGC (SEQ ID NO. 156) CAN536TTTGCGGAAGTGTCACACAAGGCGGCGCGCAGATTCAGA AGGCTGAACAAAATGATGT(SEQ ID NO. 157) CAN537 ACATCATTTTGTTCAGCCTTCTGAATCTGCGCGCCGCCTTGTGTGACACTTCCGCAAA (SEQ ID NO. 158) CAN538TGTGTGAAGCTTTTAGTGGTGGTGGTGGTGGTGGCCGCC TTTTTTATCAACTGAAAATG(SEQ ID NO. 159) CAN540 CACACACATATGCACCACCACCACCACCACAGCGCGCAGATTCAGAAGGCTGAACAAAATGATGT (SEQ ID NO. 160) CAN541CATTTTCAGTTGATAAAAAAGGCGGCACTAAAAAAGCAG CGGTATC (SEQ ID NO. 161) CAN542GATACCGCTGCTTTTTTAGTGCCGCCTTTTTTATCAACT GAAAATG (SEQ ID NO. 162) CAN543TGTGTGAAGCTTTTATTGTGTGACACTTCCGCAAA (SEQ ID NO. 163) CAN544CACACACATATGAAATACCTGCTGCCGACCGCTGCTGCTGGTCTGCTGCTCCTCGCTGCCCAGCCGGCGATGGCCCAG ATTCAGAAGGCTGAACAAAATGATGT(SEQ ID NO. 164) CAN545 GCATTTTCAGTTGATAAAAAAGGCGGCACTAAAAAAGCAGCGGTATCTG (SEQ ID NO. 165) CAN546CAGATACCGCTGCTTTTTTAGTGCCGCCTTTTTTATCA ACTGAAAATGC (SEQ ID NO. 166)CAN547 CACACACATATGAAATACCTGCTGCCGACCGCTGCTGCTGGTCTGCTGCTCCTCGCTGCCCAGCCGGCGATGGCCG ATATTCAGAAGGCTGAACAAAATGATGT(SEQ ID NO. 167) CAN548 CACACACATATGAAACACTTTCCATCCAAAGTACTGACCACAGCCATCCTTGCCACTTTCTGTAGCGGCGCACTGGCAGCCACAAACGACGACGATAAGGCTGAACAAAATGAT G (SEQ ID NO. 168) CAN670GCCGGCGATGGCCATGGATAAGGCTGAACAAAATG (SEQ ID NO. 169) CAN671CATTTTGTTCAGCCTTATCCATGGCCATCGCCGGC (SEQ ID NO. 170) CAN678GGAAGTGTCACACAATAAGGCGGCCACCACCACC (SEQ ID NO. 171) CAN679GGTGGTGGTGGCCGCCTTATTGTGTGACACTTCC (SEQ ID NO. 172) CAN1517GATATACATATGAAATACCTGCTGCCGACCGCTGCTGCTGGTCTGCTGCTCCTCGCTGCCCAGCCGGCGATGGCCA TTCAGAAGGCTGAACAAAA(SEQ ID NO. 205) CAN1518 GGCCGCAAGCTTTTAGTGGTGGTGGTGGTGGTGGCCGC C(SEQ ID NO. 206) CAN1519 GGCCGCAAGCTTTTATTGTGTGACACTTCC (SEQ ID NO. 207)CAN1523 GCTGCCCAGCCGGCGATGGCCAAGGCTGAACAAAATGA TGTG (SEQ ID NO. 208)CAN1524 CACATCATTTTGTTCAGCCTTGGCCATCGCCGGCTGGG CAGC (SEQ ID NO. 209)CAN1525 GCTGCCCAGCCGGCGATGGCCGCTGAACAAAATGATGT GAAGC (SEQ ID NO. 210)CAN1526 GCTTCACATCATTTTGTTCAGCGGCCATCGCCGGCTGG GCAGC (SEQ ID NO. 211)CAN1527 GCTGCCCAGCCGGCGATGGCCGAACAAAATGATGTGAA GCTGG (SEQ ID NO. 212)CAN1528 CCAGCTTCACATCATTTTGTTCGGCCATCGCCGGCTGG GCAGC (SEQ ID NO. 213)CAN1529 GCTGCCCAGCCGGCGATGGCCGCCCAGATTCAGAAGGC TGAAC (SEQ ID NO. 214)CAN1530 GTTCAGCCTTCTGAATCTGGGCGGCCATCGCCGGCTGG GCAGC (SEQ ID NO. 215)CAN1531 GCTGCCCAGCCGGCGATGGCCAGCGCCCAGATTCAGAA GGCTGAAC (SEQ ID NO. 216)CAN1532 GTTCAGCCTTCTGAATCTGGGCGCTGGCCATCGCCGGC TGGGCAGC (SEQ ID NO. 217)CAN1669 CACACACATATGAAATACCTGCTGCCGACC (SEQ ID NO. 218) MDesPILA-GAATTCCATATGCACCATCACCATCACCATACTAAAAA 3 AGCAGCGGTATCTGAA(SEQ ID NO. 173) MDesPILA- GCGCCGCTCGAGTCATTGTGTGACACTTCCGC 4(SEQ ID NO. 174) MnoNTHi- GCCCAGCCGGCGATGGCCCAGATCCAGAAGGCTGAACA 44AAATG (SEQ ID NO. 175) MnoNTHi- CATTTTGTTCAGCCTTCTGGATCTGGGCCATCGCCGGC45 TGGGC (SEQ ID NO. 176)Transformation

Escherichia coli BLR (DE3) or E. coli HMS (DE3) cells were transformedwith plasmid DNA according to standard methods with CaCl₂-treated cells.(Hanahan D. «Plasmid transformation by Simanis.» In Glover, D. M. (Ed),DNA cloning. IRL Press London. (1985): p. 109-135.). Briefly, BLR (DE3)or HMS174(DE3) competent cells were gently thawed on ice. Approximately4 μl of plasmid (10-100 ng) were mixed using 50-100 μl competent cells.Thereafter, this formulation was incubated on ice for 30 min. To performthe transformation reaction, the formulation was heat pulsed at 42° C.for 45 seconds then incubated on ice for 2 minutes.

Approximately 0.5 ml of SOC medium (Super Optimal broth with Cataboliterepression) was added to the transformed cells and the cell culture wasincubated at 37° C. for one hour before plating on Luria-Bertani (LB)agar with 50 ug/ml kanamycin. Around 100 μl of transformed cell culturewas plated and incubated overnight at 37° C.

BLR (DE3): BLR is a recA⁻ derivative of BL21 (F-ompT hsdSB(rB-mB-) galdcm (DE3). This E. coli strain used for expression of recombinantproteins improves plasmid monomer yields and may help stabilize targetplasmids containing repetitive sequences or whose products may cause theloss of the DE3 prophage. (Studier, F. W. (1991) J. Mol. Biol. 219:37-44). The detailed genotype of E. coli BLR (DE3) has been published byNOVAGEN®. (F-ompT hsdSB (rB-mB-) gal dcm Δ(srl-recA)306::Tn10 (TetR)(DE3).

HMS174 (DE3): HMS174 strains provide the recA mutation in a K-12background. Like BLR, these strains may stabilize certain target geneswhose products may cause the loss of the DE3 prophage. The detailedgenotype of E. coli HMS174 (DE3) has been published by NOVAGEN®.(F-recA1 hsdR(rK12-mK12+) (DE3) (Rif R).

Production Using BLR (DE3) and Characterization of His Tagged Constructsare Described in Example 3 Through Example 6

Example 3: Protein Expression Using Shake Flask

Generally, one confluent agar plate inoculated with Escherichia coli BLR(DE3) transformed with recombinant plasmid was stripped, resuspended inculture media and used to inoculate 800 ml of LB broth (Becton,Dickinson and Company)±1% (weight/volume, w/v) glucose (Laboratoire MAT,catalogue number: GR-0101) and 50 μg/ml kanamycin (Sigma) to obtainO.D._(600nm) between 0.1 and 0.2. Cultures were incubated at 37° C. withagitation of 250 RPM to reach an O.D._(600nm) of ˜0.8.

One ml of each culture was then collected, centrifuged at 14 000 RPM for5 minutes and supernatants and pellets were frozen at −20° C.separately.

At an O.D._(600nm)˜0.8, the BLR (DE3) cultures were cooled down (−20°C., 20 minutes or 4° C., 1 hour, preferably at 4° C. for 1 hour) beforeinducing the expression of the recombinant protein by addition of 1 mMisopropyl β-D-1-thiogalactopyranoside (IPTG; EMD Chemicals Inc.,catalogue number: 5815) and incubation overnight at 16, 22 and 30° C.,or 3 hours at 37° C. with agitation of 250 RPM, preferably overnight at22° C. After the induction period the cultures were centrifuged at 14000 RPM for 5 minutes or 6 000 RPM for 15 minutes and supernatant (mediafraction sample) and pellets (containing soluble and insolublefractions) were frozen at −20° C. separately.

These conditions are used for periplasmic protein expression.

Example 4: Protein Purification Using Shake Flask, Cell Pastes, HisTagged Constructs

Each bacterial pellet obtained after induction was resuspended in 20 mM4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer (pH8.0) containing 500 mM NaCl, 10 mM imidazole and Roche COMPLETE®Protease Inhibitor Cocktail (1 tablet/50 ml of HEPES buffer containing500 mM NaCl, Roche COMPLETE® ULTRA tablets, Roche DiagnosticsCorporation).

Alternatively, 20 to 50 mM bicine buffer may be used instead of HEPESbuffer containing NaCl. For example, 20 mM bicine buffer may be used.Bacteria were lysed using a Constant System 1.1 KW 2×30 000 PSI (poundsper square inch). Soluble (supernatant) and insoluble (pellet)components were separated by centrifugation at 20 000 g for 20 min at 4°C.

6-His tagged-proteins were purified under native conditions onimmobilized metal affinity chromatography (IMAC) using PROFINIA™ proteinpurification protocol (Bio-Rad Laboratories, Inc.). The solublecomponents were loaded on a 5 ml His Trap column (Bio-Rad Laboratories,Inc.) preequilibrated with the same buffer used for bacterialresuspension; the soluble components were added at up to 5 ml/min(producing a “flow through fraction”) After loading on the column, thecolumn was washed with 10 column volumes of the same buffer at a rate of10 ml/min (producing a “wash fraction #1). A second wash using 20 mMbicine buffer or 20 mM HEPES buffer (pH 8.0) containing 500 mM NaCl and20 mM imidazole was performed, producing a “wash fraction #2). Elutionwas performed using 2 column volumes of 20 mM HEPES buffer or 50 mMbicine buffer (pH 8.0) containing 500 mM NaCl and 250 mM imidazole at arate of 10 ml/min, producing an “elution fraction”.

To improve the purity of the protein, positive elution fractions fromIMAC were pooled and loaded on a size exclusion chromatography (SEC)column (HILOAD™ SUPERDEX™ 200 26/60 from GE Healthcare) preequilibratedin phosphate buffered saline without calcium or magnesium (NaCl 137 mM,KCl 2.7 mM, Na₂HPO₄ 8.1 mM, KH₂PO₄ 1.47 mM, pH 7.4). Samples fromelution fractions were analyzed by sodium dodecyl sulfate polyacrylamidegel electrophoresis (SDS-PAGE). Samples were concentrated usingCentricon 10 000 MW (Millipore).

Protein concentration was determined using spectrometer.

Example 5: SDS-PAGE and Western Blot Analysis of His Tagged Constructs &SDS-PAGE Analysis of Non-His Tagged LVL317 & LVL318 Constructs

Soluble and Insoluble Fraction Preparation

For example, 1 ml of culture after induction (see, for example, Example3 above) was centrifuged at 14 000 RPM for 2 min. The pellet wasresolubilized using 40 μl of BUGBUSTER® Protein Extraction Reagent(NOVAGEN®, EMD4 Biosciences, Merck), creating a cell suspension. Thecell suspension was incubated on a rotating platform for 10 min at roomtemperature. The cell suspension was then centrifuged at 14 000 RPM for2 min to separate the soluble fraction. The resulting pellet (insolublefraction) was resolubilized using 70 μl of deionized water, 5 μl ofdithiothreitol (DTT) 1M and 25 μl of NUPAGE® LDS (Lithium DodecylSulphate) Sample Buffer 4× (INVITROGEN™). The soluble fraction(supernatant from the cell suspension of the resolubilized pellet) wasadded to 30 μl of deionized water, 5 μl of DTT 1M and 25 μl of LDSSample Buffer 4×.

Media Fraction Preparation

For example, to prepare the media fraction, 100 μl of the supernatantfrom the induced whole cell culture following centrifugation (see, forexample, Example 3 above) was concentrated by adding 500 μl of RCreagent I (Bio-Rad Laboratories, Inc.); the sample was mixed andincubated for 1 min at room temperature. Then, 500 μl of Reagent II(Bio-Rad Laboratories, Inc.) was added to the sample and mixed. Thisformulation was centrifuged at 14 000 RPM for 10 min. The pellet wasresolubilized using 28 μl of deionized water, 2 μl of DTT 1M and 10 μlof LDS SB 4×.

Purification Fraction Preparation

For example, purified proteins (for example, obtained as described inExample 4) were prepared for SDS-PAGE analysis by adding 70 μl ofsample, 5 μl of DTT 1M and 25 μl of LDS Sample Buffer 4×.

SDS-PAGE Analysis and Transfer to Nitrocellulose Membrane

SDS-PAGE analysis and transfer to nitrocellulose membrane were performedaccording to manufacturer's recommendations (Invitrogen) using NUPAGE®Bis-Tris 4-12% gels. Preparations of samples, buffers and migrationconditions were done under conditions recommended by the suppliers.

In one example, the gel was loaded with a 20 ul sample from a master mixcomprising 70 μl of a purified protein fraction, 5 μl of DTT 1M and 25μl of LDS SB 4×.

After samples were run on NUPAGE® Bis-Tris 4-12% gels, the proteins weretransferred to nitrocellulose membranes.

Nitrocellulose membranes were blocked for 30 minutes at 37° C., 60 RPMusing 3% milk/PBS 1× fresh solution. After the blocking incubation,Primary Antibodies were added (6× His Tag® antibody, Abcam PLC,catalogue number: ab9108) at a dilution of: 1:1000 in 3% milk/PBS 1×fresh solution for 1 hour at 37° C., 60 RPM. After that, membranes werewashed three times, for 5 minutes each, at room temperature using 0.02%polsorbate 20 (for example, TWEEN™ 20)/PBS 1×. Secondary Antibodies(alkaline phosphatase (AP) Rabbit anti-IgG (H+L) rabbit, JacksonImmunoResearch Laboratories, Inc.) were added at dilution 1:14 000 using3% milk/PBS 1× fresh solution. Membranes were incubated for 1 hour at37° C., 60 RPM. After that, membranes were washed three times for 5minutes at room temperature using 0.02% polysorbate 20 (for example,TWEEN™ 20)/PBS 1× before the membrane expositions to5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (forexample, BCIP®/NBT from Sigma-Aldrich®, 1 tablet/10 ml water).

See FIG. 1 for SDS-PAGE of induced bacterial extracts for fusion proteinconstructs LVL291, LVL268 and LVL269. Insoluble fraction (I), Solublefraction (S) and Culture Media fraction (M) were loaded for LVL291,LVL268 and LVL269 before and after induction (ind).

See FIG. 2 for SDS-PAGE and Western blot related to purificationextracts for fusion protein constructs LVL291, LVL268 and LVL269. Flowthrough fraction (Ft), Wash fraction (W) and Elution fraction (E) wereloaded for purification of LVL291, LVL268 and LVL269. Anti-his tag wasused to probe extracts.

See FIG. 3 for SDS-PAGE of induced bacterial and purification extractsfor fusion protein constructs LVL291 and LVL315. Culture Media fraction(M), Soluble fraction (Sol), Insoluble fraction (Ins), Flow throughfraction (Ft), Wash fraction #1 (W1), Wash fraction #2 (W2) and Elutionfraction (E) were loaded for LVL291 and LVL315.

See FIG. 4 for SDS-PAGE of induced bacterial and purification extractsfor fusion protein construct LVL312. Culture Media fraction (M), Solublefraction (Sol), Insoluble fraction (Ins), Flow Through fraction (Ft),Wash fraction #1 (W1), Wash fraction #2 (W2) and Elution fraction (E)were loaded for LVL312.

See FIG. 25 for SDS-PAGE of soluble fractions from induced bacterialextracts for fusion protein constructs LVL291, LVL702, LVL736, LVL737,LVL738, LVL739, LVL740 and pET26b vector (negative control). (a)Experiment 1 (b) Experiment 2 (c) Experiment 3. PE-PilA fusion proteinindicated by arrow.

See FIG. 26 for the average band percentage of fusion protein in thesoluble fraction from Experiments 1, 2 and 3.

LVL317 and LVL318 bacterial extracts used in the SDS-PAGE analysis inFIG. 5 and FIG. 6, respectively, were prepared generally as describedabove.

FIG. 5. SDS-PAGE of induced (1 mM and 10 μM IPTG) bacterial extracts forfusion protein construct LVL317. Extracts from before (NI) and afterinduction (In), Soluble fraction (S), Insoluble fraction (I).

FIG. 6. SDS-PAGE of induced (1 mM and 10 μM IPTG) bacterial extracts forfusion protein construct LVL318. Extracts from before (NI) and afterinduction (In), Culture Media fraction (M), Soluble fraction (S),Insoluble fraction (I).

Proteins separate by SDS-PAGE were transferred to an Immobilon-Pmembrane. The Coomassie Blue stained protein bands were cut and placedin a sequenator reactor. Sequencing was carried out according tomanufacturer's protocol using an Applied Biosystems PROCISE® ProteinSequencer, model 494-cLC.

TABLE 5 Shake flask protein expression profiles and signalpeptide cleavage for fusion protein constructs. Fusion Protein SignalProtein Expression peptide Construct ID Description N-term → C-termprofile cleavage LVL312 Flgl sp-E-PilA fragment-GG-PE fragment- In: +++Confirmed GGHHHHHH So: + Se: + LVL291PelB sp-PE fragment-GG-PilA fragment- In: +++ Confirmed GGHHHHHH So: ++Se: + LVL268 PelB sp-D-PE fragment-GG-PilA fragment- In: +++ ConfirmedGGHHHHHH So: ++ Se: + LVL269 NadA sp-ATNDDD-PE fragment-GG-PilA In: +++Confirmed fragment-GGHHHHHH So: ++ Se: + LVL270MHHHHHH-PE fragment-GG-PilA fragmnet In: +++ Not  So: - tested Se: -LVL315 PelB sp-MD-PE fragment-GG-PilA fragment- In: +++ ConfirmedGGHHHHHH So: ++ Se: + LVL317 PelB-PE fragment-GG-PilA fragment In: +++Confirmed So: + Se: Nt LVL318 PelB sp-MD-PE fragment-GG-PilA fragmentIn: +++ So: + Se: − LVL702 PelB sp-PE fragment-GG-PilA fragment- In: +++Confirmed GGHHHHHH So: ++ Se: Nt LVL736PelB sp-PE fragment-GG-PilA fragment- In: +++ Confirmed GGHHHHHH So: ++Se: Nt LVL737 PelB sp-PE fragment-GG-PilA fragment- In: +++ ConfirmedGGHHHHHH So: ++ Se: Nt LVL738 PelB sp-PE fragment-GG-PilA fragment-In: +++ Confirmed GGHHHHHH So: ++ Se: Nt LVL739PelB sp-PE fragment-GG-PilA fragment- In: +++ Confirmed GGHHHHHH So: ++Se: Nt LVL740 PelB sp-PE fragment-GG-PilA fragment- In: +++ ConfirmedGGHHHHHH So: ++ Se: Nt So = Soluble fraction. In = Insoluble fraction.Se = Protein Secreted in the media fraction. Nt = Not tested. Thefollowing rating were based on a visual inspection (coomassie) +: lowexpression; ++: medium expression; +++: high expression; −: noexpression

Example 6: LVL291 Fusion Protein Characterization

Physical Properties of LVL291: Folding of PE and PilA in LVL291 &Melting Point Circular Dichromism:

Analysis of Secondary Structure

Circular dichroism (CD) is used to determine the secondary structurecomposition of a protein by measuring the difference in the absorptionof left-handed polarized light versus right-handed polarized light whichis due to structural asymmetry. The shape and the magnitude of the CDspectra in the far-UV region (190-250 nm) are different whether aprotein exhibits a beta-sheet, alpha-helix or random coil structure. Therelative abundance of each secondary structure type in a given proteinsample can be calculated by comparison to reference spectra.

Far UV spectra are measured using an optical path of 0.01 cm from 178 to250 nm, with a 1 nm resolution and bandwidth on a Jasco J-720spectropolarimeter. Temperature of the cell is maintained at 23° C. by aPeltier thermostated RTE-111 cell block. A nitrogen flow of 10 L/min ismaintained during the measurements.

Results:

The far-UV CD spectra obtained for PE (from construct pRIT16762), PilA(from construct pRIT 16790) and PE-PilA proteins are characteristic offolded proteins containing a mix of alpha and beta structures, but PE issignificantly richer in alpha helix than PilA and PE-PilA (FIG. 7, CDspectra of PE, PilA and PE-PilA fusion proteins).

In order to evaluate the integrity of the folding of PE and PilAindividual proteins once bound together in a chimeric protein and thenverify a possible interaction between both, difference spectra werecalculated.

-   -   When the PE and PilA far-UV spectra are combined, the resulting        spectrum superposes to the spectrum of PE-PilA chimer (FIG. 8,        Combination of PE and PilA CD spectrum). This result suggests        that the PE-PilA chimer contains all the secondary structures        that are detected in the individual components. It also suggests        that the fusion of the proteins has no major impact on the        secondary structures of the individual components and        consequently that the folding of PE and PilA is not        significantly different whether the proteins are separate or in        fusion.        Melting Point Evaluation:

In order to evaluate if the expression in fusion has an impact on thethermodynamic properties of the individual proteins, the melting pointsof PE, PilA and PE-PilA have been evaluated by monitoring the defoldingof the alpha helix with temperature by circular dichroism.

The presence of alpha helix is characterized by a minimum in theCircular dichroism signal at 222 nm, so a significant increase in CDsignal at 222 nm during temperature increase is an indication of proteindenaturation. The determination of the temperature at which the proteinundergoes loss in secondary structure allows the determination of themelting point (Tm), which corresponds to the temperature at which halfof the proteins have lost their structure.

Melting point can be determined by identification of the inflexion pointon the thermal denaturation curve obtained from a temperature versus CD222 nm plot.

-   -   Melting point of PilA and PE as determined by far-UV CD are        respectively of 52° C. and 68° C. (FIG. 9, PilA thermal        denaturation curve; FIG. 10, PE thermal denaturation curve).    -   The PE-PilA fusion protein exhibits two distinct Tm's at 48° C.        and 71° C. (FIG. 11, PE-PilA fusion protein thermal denaturation        curve). Those values indicate that the PE and PilA proteins are        still independently folded when bound into a chimer and that        they defold at a similar temperature whether they are separate        or in fusion. The observation that the defolding of the PilA        portion at 48° C. doesn't cause precipitation or impact the Tm        of the PE portion at 71° C. is a strong indication that the        interaction between PE and PilA within the fusion is minimal and        that they don't have a major observable impact on each other.        The melting points of proteins are sensitive to various external        conditions, including buffer composition or presence of        interacting molecules; that no major variation is observed upon        fusion of PE and PilA is a strong indication of the preservation        of most of the structure and of the properties of both PE and        PilA when they are bound together.

Example 7: Fermentation Process

Fusion proteins of the invention may be prepared by methods known bythose skilled in the art.

Example 8: Protein Purification of PE, PilA, and LVL317

PE Protein Purification from pRIT16762:

To generate the pRIT16762 expression vector, the pRIT16711 vector wasdigested using BamHI and NcoI restriction enzymes in order to delete 6amino acid residues between the signal sequence (pelB) and PE. Thevector obtained was named pRIT16712. In this vector, there are 3 aminoacids between the signal sequence pelB and PE: MDP. In a second step, asite directed mutagenesis was performed to change amino acid sequencefrom MDP to QIQ using pRIT16712 as template with primers MnoNTHi-44 andMnoNTHi-45 (described in Table 4) and the QuikChange II Site-DirectedMutagenesis Kit (Agilent Technologies, Stratagene Division).

Working seed of E. coli BLR(DE3) containing PE QIQ (from the pRIT16762construct) was thawed from −80° C. and used to prepare 100 ml ofpre-culture in LB broth by overnight incubation at 37° C. underagitation at 215 RPM. After overnight incubation, eight flaskscontaining 800 ml of LB APS were inoculated with 12.5 ml of pre-cultureand OD₆₀₀ measured at around 0.06. The cultures were incubated 3 h at37° C. with shaking. At a OD₆₀₀ of around 0.9, 1 mM IPTG was added tostart the induction. During the induction, the cultures were incubated19 h at 22° C. with shaking. After induction, OD₆₀₀ was at around 2.2.The cell cultures were transferred into 1 L centrifuge bags placedinside 1 L bottles and centrifuged at 4° C. for 30 minutes at 6,000×gand supernatant discarded. 1 ml aliquots of culture pre- andpost-induction and supernatant were kept for future analysis.

Lysis of the BLR(DE3) Induced with PE Q/Q

The centrifuge bags were removed from the centrifugation bottles, openedand the pellet was expulsed from the bag into a beaker. The eightpellets were pulled together and resuspended in 100 ml of binding buffer(20 mM Hepes, 10 mM imidazole, 500 mM NaCl, pH 8.01). The E. coli BLR(DE3) containing the PE QIQ construct were disrupted with the TS SeriesBench Top cell disrupter from Constant Systems Ltd. (1×30 kPsi; 1×15kPsi). The lysate was centrifuged 30 minutes, 6000 RPM, 4° C. Thesupernatant was kept and loaded on an IMAC column.

IMAC Purification of PE Q/Q

IMAC column (BioRad, Bio-Scale Mini Profinity IMAC cartridge 5 ml) wasequilibrated with 5CV of Binding buffer (20 mM HEPES, 10 mM imidazole,500 mM NaCl, pH 8.01) at 5 ml/min. 100 ml of lysate supernatant wasloaded on the IMAC at 2.5 mL/min. Flow-through was collected in 50 mlfractions for future analysis. The column was washed with 3CV of Bindingbuffer to remove unbound protein. Sample containing unbound proteins wascollected in one aliquot of 15 ml in a 50 ml tube. The column was washedwith 2CV of Wash buffer (20 mM HEPES, 20 mM imidazole, 500 mM NaCl, pH8.01) collected in 2 ml fractions in a 96 well plate. The bound proteinwas then eluted with 6CV of 100% Elution buffer (20 mM HEPES, 250 mMimidazole, 500 mM NaCl, pH 8.01). The eluted protein was collected in 2ml fractions in 96-well plates. Wash and elution were performed at 5ml/min.

Size Exclusion Chromatography (SEC) on the IMAC Pool of PE Q/Q

SEC column (GE healthcare, HILOAD™ 26/60 SUPERDEX™ 75 prep grade, 60 cmheight approx 319 ml volume) was equilibrated with 3CV of SEC buffer (20mM HEPES, 150 mM NaCl, pH8.49). 11 ml of IMAC eluate was loaded onto thecolumn at a flow rate of 2.5 ml/min. 2 ml fractions were collected from0.3CV to 0.9CV. Two runs were performed then fractions were analyzed bySDS-PAGE. Fractions from the two runs containing Prot E protein werepooled together (“SEC pool”, 48 ml approx total volume). 500 mM ofArginine was added to the SEC pool.

Dosage of the PE QIQ Pooled Samples Generated in the Above SEC Protocol

The SEC pool was dosed with the RCDC (Reducing Agent and DetergentCompatible) method from the Bio-Rad RC DC™ kit following manufacturer'sprotocol:

For each tested sample and standard, 25 μL was distributed in microfugetubes in duplicate. 125 μL of Bio-Rad RC Reagent I was added into eachtube; each tube was vortexed and incubate for 1 minute at roomtemperature. 125 μL of Bio-Rad RC Reagent II is added into each tube;each tube is vortexed and then centrifuged at 14,000×g for 5 minutes.Supernatants are discarded by inverting the tubes on clean, adsorbenttissue paper allowing the liquid to drain completely from the tubes.25.4 μL of Reagent A (already prepared by mixing 20 μL of Reagent S per1 ml of Reagent A) is added to each tube; each tube is vortexed andincubated at room temperature for 5 minutes, or until precipitate iscompletely dissolved. Vortex before proceeding to next step. Add 200 μLof DC reagent B to each tube and vortex immediately. Incubate at roomtemperature for 15 minutes. Transfer all samples to a 96-well plate andread the adsorbance at 750 nm to determine the protein concentration foreach unknown protein sample.

The ProtE concentration was 1.069 mg/ml

PilA His-Tagged Protein Purification:

PilA was purified following the general procedure below:

E. coli cells containing a construct encoding PilA or a fragment thereofare suspended in BUGBUSTER® and BENZONASE® nuclease (NOVAGEN®), forexample 10 ml BUGBUSTER® and 10 ul BENZONASE® nuclease. The cell lysateis mixed at room temperature on a rotating platform, for example, for 15minutes. The cell lysate is centrifuged at 4° C., for example at 16,000g for 20 minutes. The supernatant containing the protein is added to aNi NTA column containing Ni NTA HIS⋅BIND® resin and mixed at 4° C., forexample for 1 hour. The column may consist of 2 ml of Ni NTA HIS⋅BIND®resin (NOVAGEN®) and 10 ml 1× Binding Buffer (from NOVAGEN®'s Ni-NTABuffer Kit). The column flow through is then collected. The resin iswashed two times with 1× wash buffer, for example, containing 300 mMNaCl, 50 mM NaH₂PO₄, 25 mM imidazone, pH 8.0). The wash is collected bygravity flow. The protein is eluted from the column with 1× elutionbuffer, for example, 300 mM NaCl, 50 mM NaH₂PO₄, 250 mM imidazone, pH8.0. The protein may be further purified by dialysis with the BindingBuffer and rerun over a Ni NTA column as described above.

Thrombin Cleavage of PilA.

PilA is then incubated with thrombin (diluted 1/50) at room temperaturefor 16 h, to remove the histidine tag.

Size Exclusion Chromatography (SEC) on PilA Cleaved with Thrombin.

SEC column (GE healthcare, HILOAD™ 26/60 SUPERDEX™ 75 prep grade, 60 cmheight approx 319 ml volume) was equilibrated with 5CV of SEC buffer (20mM HEPES, 150 mM NaCl, pH8.52). Approximately 10 ml of cleaved PilA wasloaded onto the column at a flow rate of 2.5 ml/min. 2 ml fractionscollected from 0.3CV to 0.9CV. Two runs were performed then fractionswere analyzed by SDS-PAGE. Fractions from the two runs containingcleaved PilA protein were pooled together (“SEC pool”, 52 ml approxtotal volume).

Dosage of PilA, SEC Pool.

The SEC pool was dosed with the RCDC method as described above. Thecleaved PilA concentration was at 5.37 mg/ml.

Dialysis of the PilA SEC Pool with PBS Lx pH 7.4 (Dialysis Factor=1600)and Dosage by RCDC

The concentration post-dialysis determined by RCDC was at 3.0 mg/ml.

Purification of LVL317

Osmotic Shock

Since LVL317 fusion protein is expressed and processed in bacterialperiplasm, the protein was extracted by osmotic shock.

Frozen (−20° C.) harvested E. coli B2448 cell paste containing LVL317from 4 L of fermentor culture were pooled and resuspended in ahypertonic buffer consisting of 24 mM Tris-HCl, 16% (w/v) sucrose, 9.9%(w/v) glucose, 10 mM EDTA, pH 8.0 up to a final volume of 4 L. Thesuspension was mixed gently for 30 min at room temperature using a3-blade propeller installed on RW 16 basic stirrer, at medium speed. Thesuspension was centrifuged at 15,900×g for 30 minutes at roomtemperature. Supernatant (SN1) was kept for gel analysis.

The resulting pellet was resuspended in a hypotonic solution; 38 mMMgCl₂, and mixed for 30 min at room temperature. The mixture wascentrifuged at 15,900×g for 30 minutes at room temperature and theantigen recovered in the supernatant (SN2).

A clarification of the SN2 was performed by filtration through a0.45/0.2 μm polyethersulfone Sartorius Sartopore 2 MidiCap filter, at600 ml/min of flow rate.

The SN2 was diluted 1:3 with 20 mM NaH₂PO₄—Na₂HPO₄, pH 7.0, the pHadjusted to 7.0 if necessary and another clarification by filtrationthrough a 0.45/0.2 μm polyethersulfone Sartorius Sartopore 2 MidiCapfilter, at 600 ml/min was performed.

SP SEPHAROSE™ Fast Flow (SP FF) Chromatography

The diluted/filtered SN2 was loaded and captured on a strong cationicexchanger resin (SP SEPHAROSE™ FF—GE Healthcare) in a 14 cm ID (internaldiameter)×20 cm length column (column volume 3100 ml) equilibrated with2CV of 20 mM NaH₂PO₄/Na₂HPO₄ buffer pH 7.0. After washing the columnwith 5CV of 20 mM NaH₂PO₄/Na₂HPO₄ buffer pH 7.0, the antigen (containedwithin LVL317) was eluted by increasing the concentration of NaCl up to100 mM in the same washing buffer.

See FIG. 12 for a typical SP SEPHAROSE™ Fast Flow chromatogram.

Q SEPHAROSE™ Fast Flow (Q FF) Chromatography

The antigen present in the SP FF Eluate was diluted 1:4 with a 20 mMTris pH 8.5, pH adjusted to 8.5 if necessary and passed through a stronganionic exchanger resin (Q SEPHAROSE™ FF—GE Healthcare) in a 14 cmID×11.8 cm length column (column volume 1800 ml) equilibrated with 2CVof 20 mM Tris buffer pH 8.5. The antigen was recovered in theflow-through fraction.

See FIG. 13 for a typical Q SEPHAROSE™ Fast Flow chromatogram.

Concentration, Diaflitration, Polysorbate 80 Addition and SterileFiltration

The Q FF flow-through containing the antigen was concentrated up to0.7-0.8 mg/ml based on chromatogram UV and diafiltered with 5DV of 10 mMKH₂PO₄/K₂HPO₄ buffer pH 6.5 using a Pellicon-2™ 10 kDa cutoff membrane(Millipore).

Using a 5% stock solution, polysorbate 80 (for example, TWEEN™ 80) wasadded to the ultrafiltration retentate and agitated for 30 minutes withmagnetic stirrer at 130 rpm at 4° C. The final concentration ofpolysorbate 80 was 0.04%. Ultrafiltration retentate was sterilized byfiltration through a 0.45/0.2 μm Cellulose Acetate membrane (Sartobran300, Sartorius). The purified bulk was stored at −20° C. or −80° C.Absolute protein concentration was measured by AAA (Amino Acid Analysis)at 0.737 mg/ml.

Example 9: Use of Polysorbate 80

A titration experiment indicated that the addition of polysorbate 80,specifically, TWEEN™ 80 to a final concentration of 0.04% (w/v) to thepurified bulk prior to sterile filtration reduced filamentous particleformation and aggregation.

According to DSC analysis, TWEEN™ 80 reduced the degree of structuralchange (30-45° C.) seen after freeze/thaw cycles after storage at −20°C. and after storage 4 days at 4° C., −20° C. and −80° C. and 37° C.

Example 10: SDS-PAGE and Western Blot Analysis of LVL317

SDS-PAGE and Western Blot Analysis:

NUPAGE®, Bis-Tris 4-12% gel was loaded as described below with 10 μg ofsample in NUPAGE® LDS sample buffer containing 50 mM DTT heated 5 min at95° C. (20 μL of sample was loaded for samples having lowconcentration). Migration: 35 minutes at 200 Volts at room temperature(RT) in NUPAGE® MES Running Buffer. Gel Stained 2 hours in Instant blue(Novexin cat.: ISB01 L) and destained overnight in water.

Lane Contents:

 1: MW standard (10 μL)  2: Start (total fraction) (10 μg)  3: SN1 nonfiltered (10 μg)  4: SN2 not filtered (10 μg)  5: Not extracted (10 μg) 6: Load SP FF (10 μg)  7: Flow through SP FF (6.9 μg)  8: Wash SP FF(20 μL)  9: Elution SP FF (10 μg) 10: Strip SP FF (10 μg) 11: Load Q FF(8.9 μg) 12: Elution Q FF (9.8 μg) 13: Strip Q FF (4.8 μg) 14: TFFretentate before0.04% TWEEN ™ 80 spiked (10 μg) 15: Purified bulk Notfiltered 0.04% TWEEN ™ 80 spiked (10 μg) 16: Purified bulk SterileFiltered 0.04% TWEEN ™ 80 spiked (10 μg) 17: Purified bulk SterileFiltered 0.04% TWEEN ™ 80 spiked (20 μg + spiked E. Coli Cell lysate  Rix (1 μg)) 18: E. Coli Cell lysate Rix (2 μg) 19: E. Coli Cell lysateRix (1 μg) 20: E. Coli Cell lysate Rix (0.5 μg)

See FIG. 14 for a SDS-PAGE of In-process samples from purificationprocess of PE-PilA fusion protein.

For Western Blot, proteins were transferred at 4° C. overnight at 30Volts in NUPAGE® transfer buffer+20% Methanol, 0.1% SDS onnitrocellulose membrane. Membranes were blocked 1 hour with 50 mM Tris,150 mM NaCl pH 7.4+5% non-fat dry milk, incubated 2 hours in rabbitpolyclonal primary antibody diluted in blocking buffer (anti-Prot-E 1/50000 and anti-E. coli (BLR) 1/1 000), washed 3×5 minutes in 50 mM Tris pH7.4+0.05% Tween 20, incubated 1 hour in secondary antibody (goatanti-rabbit conjugated to alkaline phosphatase diluted 1/5000 inblocking buffer), washed 3×5 minutes in wash buffer and developed inBCIP/NBT substrate (1 tablet per 10 ml). All incubations performed in 25ml per membrane.

See FIG. 15 for a Western Blot of In-process samples of purificationprocess from PE-PilA fusion protein. Blot using rabbit polyclonalanti-PE.

Lane Contents:

 1: MW standard (10 μL)  2: Start (total fraction) (10 μg)  3: SN1 nonfiltered (10 μg)  4: SN2 not filtered (10 μg)  5: Not extracted (10 μg) 6: Load SP FF (10 μg)  7: Flow through SP FF (6.9 μg)  8: Wash SP FF(20 μL)  9: Elution SP FF (10 μg) 10: Strip SP FF (10 μg) 11: Load Q FF(8.9 μg) 12: Elution Q FF (9.8 μg) 13: Strip Q FF (4.8 μg) 14: TFFretentate before0.04% TWEEN ™ 80 spiked (10 μg) 15: Purified bulk Notfiltered 0.04% TWEEN ™ 80 spiked (10 μg) 16: Purified bulk SterileFiltered 0.04% TWEEN ™ 80 spiked (10 μg) 17: Purified bulk SterileFiltered 0.04% TWEEN ™ 80 spiked (20 μg + spiked E. Coli Cell lysate  Rix (1 μg)) 18: E. Coli Cell lysate Rix (2 μg) 19: E. Coli Cell lysateRix (1 μg) 20: E. Coli Cell lysate Rix (0.5 μg)

See FIG. 16 for a Western Blot of In-process samples of purificationprocess from PE-PilA fusion protein. Blot using rabbit polyclonalanti-E. coli (BLR).

Lane Contents:

 1: MW standard (10 μL)  2: Start (total fraction) (10 μg)  3: SN1 nonfiltered (10 μg)  4: SN2 not filtered (10 μg)  5: Not extracted (10 μg) 6: Load SP FF (10 μg)  7: Flow through SP FF (6.9 μg)  8: Wash SP FF(20 μL)  9: Elution SP FF (10 μg) 10: Strip SP FF (10 μg) 11: Load Q FF(8.9 μg) 12: Elution Q FF (9.8 μg) 13: Strip Q FF (4.8 μg) 14: TFFretentate before0.04% TWEEN ™ 80 spiked (10 μg) 15: Purified bulk Notfiltered 0.04% TWEEN ™ 80 spiked (10 μg) 16: Purified bulk SterileFiltered 0.04% TWEEN ™ 80 spiked (10 μg) 17: Purified bulk SterileFiltered 0.04% TWEEN ™ 80 spiked (20 μg + spiked E. Coli Cell lysate  Rix (1 μg)) 18: E. Coli Cell lysate Rix (2 μg) 19: E. Coli Cell lysateRix (1 μg) 20: E. Coli Cell lysate Rix (0.5 μg)SDS-PAGE and Western Blot Figures Comments:

The PE-PilA fusion protein migrates at 30 kDa. The extraction by osmoticshock extracts the fusion protein expressed and processed in bacteriaperiplasm and reduced contamination from bacteria. Small loss of fusionprotein during hypertonic treatment (lane 3). A small proportion is notextracted by hypotonic treatment and remains associated with cells (lane5). Small loss in SP FF Flow through (lane 7) and in strip fraction ofboth columns (lanes 10 and 13). Since the total volume of strip fractionis low the loss of fusion protein is not significant. Degraded bands arevisible in strip fractions but not in final product. No significantcontamination from E. coli host cell proteins in purified bulk (lane16).

Analysis of LVL735 and LVL778 yielded similar profiles as LVL317.

Example 11: Melting Point Data for PE, PilA and LVL317

Thermal transition of PE-PilA fusion non His-tagged protein (LVL317) wascompared with the thermal transition of both PE his-tagged (as describedin Example 8) and cleaved PilA (as described in Example 8) proteins,purified as described above.

Before DSC, PE and PilA were dialyzed overnight in 10 mM K₂HPO₄/KH₂PO₄pH 6.5+0.04% Tween 80 (1:250 sample:buffer volume ratio) to have them inthe same buffer as the fusion protein. After dialysis, proteinsconcentration was measured by BCA and adjusted to 300 μg/ml (PE) and 500μg/ml (PilA).

Analysis done on VP™-DSC from MicroCal, LLC (part of GE Healthcare). Thefinal dialysis buffer was used as reference and subtracted from thescans. DSC scan rate 90° C./hr. In order to evaluate the capacity tomeasure the thermal transition in the Final Container (FC) afterformulation, the fusion protein was diluted to the FC concentration (60μg/ml). Final container data not shown.

Results:

See FIG. 17 for Thermal transition of PE-PilA fusion protein and PE andPilA proteins. Curves: PilA (1), Protein E (Prot E, PE) (2), PE-PilA PBnot diluted 737 μg/ml (3), and PE-PilA PB diluted at FC concentration 60μg/ml (4).

1 - PilA Tm: 53° C. 2 - Protein E Tm: 63 3 - PE-PilA PB (Purified Bulk)not Tm₁: 53.7° C. and Tm₂: 66.1° C. diluted 737 μg/ml 4 - PE-PilA PBdiluted at FC Tm1: 53.2° C. and Tm2: 67.6° C. concentration 60 μg/ml

Two transitions were detected in the purified fusion protein (LVL317)(curves 3 and 4).

The Tm₁ (53.7° C.) of the PE-PilA fusion protein is similar to PilAtransition (53° C.).

Significant shift of Tm₂ in PE-PilA (66.1° C.) as compared to PEtransition (63° C.). The fusion of both domains seems to stabilize thePE fragment.

The shift of Tm₂ in the diluted fusion protein as compared to undilutedis a concentration artifact arising from the steep decreasing slopetypical of aggregation which is concentration dependant.

Antigen folding analysis of LVL735 and LVL778 were similar to that ofLVL317.

Example 12: PE-PilA Fusion Protein Construct LVL291 Anti-PilAImmunogenicity Response in Balb/c Mice

The immune response directed against purified LVL291 PE-PilA fusionprotein (the LVL291 fusion protein without the heterologous signalpeptide) formulated in AS03_(A) was evaluated in Balb/c mice. Animals(20 mice/group) were immunized by the intramuscular route at days 0, 14and 28 with 10 μg of PE (from vector pRIT16762), PilA (from vectorpRIT16790) or PE-PilA, each formulated in AS03_(A). The control groupwas vaccinated with AS03_(A) alone. Antibody response directed againsteach antigen was determined in individual sera collected at day 42. Noantibody response was obtained with the negative control. As shown inFIG. 18, the antibody response directed against PilA was higher in miceimmunized with the PE-PilA fusion compared to antibody response in miceimmunized with monovalent PilA. The antibody responses directed againstPE were similar in mice immunized with the fusion protein and miceimmunized with monovalent PE. GMT=geometric means titer. Data werecaptured and analyzed with the SOFTMAX® Pro Software (Molecular Devices)running under WINDOWS® (Microsoft); the four parameters logistic logfunction was used to calculate the standard curve. The four-parameterlogistic-log function describes, with a high degree of accuracy, thecurve of the reference serum displaying a pronounced sigmoidal shapewhen plotted on an optical density-versus-concentration (log) scale.Antibody concentrations were calculated at each dilution of mice serumsamples by interpolation of the standard curve. The antibody in qualitycontrol sera and in unknown serum samples is obtained by averaging thevalues from all dilutions that fall within the working range (10-80%) ofthe dilution curve of the reference.

Results are shown in FIG. 18, which graphs the antibody responsesagainst LVL291 PE-PilA fusion protein and against monovalent PE and PilAin the Balb/c mouse model.

Example 13: Murine Nasopharyngeal Colonization Model. Immunization withPE-PilA. Challenge with NTHi Strain 86-028NP and NTHi Strain 3224A

Balb/c female mice (20/group) were immunized intranasally at days 0 and14 with 6 μg of a purified PE-PilA fusion protein (LVL291 for challengewith 86-028NP; LVL317 for challenge with strain 3224Δ) formulated withLT (heat labile toxin of Escheria coli) and on day 28 with 6 μg of apurified PE-PilA fusion protein in phosphate buffered saline (PBS).Control mice (20/group) were vaccinated with LT alone. Mice weresubsequently challenged intranasally with 5×10⁶ CFU (colony formingunits) of homologous NTHi strain 86-028NP and heterologous NTHi strain3224Δ. Homology and heterology are determined by reference to the NTHistrain with which the mice were immunized. Bacterial colonies werecounted in nasal cavities removed 1 and 2 days after the challenge.D1=day 1. D2=day 2.

PE-PilA vaccination increased the clearance of NTHi strain 86-028NP andstrain 3224A in the nasopharynx at day 1 and day 2 post challenge.

For the experiment performed with NTHi strain 86-028NP: A 2-way fixedANOVA was performed using the log 10 values of the counts as response,the fixed factors being the group (4 levels) and the day (2 levels). Theassumption of variance heterogeneity was rejected and a model withheterogeneous variances was fitted to the data. No significantinteraction was detected between the 2 factors. The group fusion PE-PilA(6 μg per mouse) significantly reduced CFU compared with the controlgroup (LT); the geometric mean ratio being equal to 0.06 with a 95%confidence interval of 0.01, 0.25.

For the experiment conducted with NTHi strain 3224Δ: A 3-way fixed ANOVAwas performed using the log 10 values as response, the fixed factorsbeing the group, the day, and the experiment. The Shapiro-Wilk andLevene's test did not reject the assumptions of normality and ofhomogeneity of variances. No significant interaction between any of the2 factors or between the 3 factors was detected and only main factorswere kept in the analysis. PE-PilA/LT significantly reduced CFU comparedwith the control group; the geometric mean ratio being equal to 0.11with a 95% confidence interval of 0.02, 0.61.

See FIG. 19 for effect of PE-PilA fusion protein vaccination on NTHistrain 86-028NP bacterial clearance in mouse nasopharynx.

See FIG. 20 for effect of PE-PilA fusion protein vaccination on NTHistrain 3224A bacterial clearance in mouse nasopharynx.

Example 14: Murine Nasopharyngeal Colonization Model. Immunization withPilA. Challenge with NTHi Strain 3219C

Female OF1 mice (20 mice/group) were immunized intranasally at days 0and 14 with 3 μg PilA (from vector 16790) formulated with LT and at day28 with 3 μg PilA in PBS. Control mice were vaccinated with LT alone.Mice were subsequently challenged intranasally with 5×10⁶ CFU of NTHistrain 3219C. Bacterial colonies were counted in nasal cavities removed3 and 4 days after the challenge. D3=day 3. D4=day 4.

See FIG. 21 for effect of PilA vaccination on bacterial clearance inmouse nasopharynx.

Example 15: Murine Nasopharyngeal Colonization Model. Immunization withPE. Challenge with NTHi Strain 3224A

Balb/c female mice (20 mice/group) were immunized intranasally at days 0and 14 with 3 μg PE (from vector pRIT16762) formulated with LT and atday 28 with 3 μg PE in PBS. Control mice were vaccinated with LT alone.Mice were subsequently challenged intranasally with 5×10⁶ CFU of NTHistrain 3224Δ. Bacterial colonies were counted in nasal cavities removed3 and 4 days after the challenge. 10 mice were examined on day 3 (D3).10 mice were examined on day 4 (D4). PE vaccination increasedsignificantly the clearance of NTHi in the naso-pharynx at day 4 postchallenge (FIG. 22), using on the Dunn test for statistical analysis.

See FIG. 22 for effect of PE vaccination on bacterial clearance in thenasopharynx of mice.

Example 16: Vibronectin Binding. Inhibition of Vibronectin Binding byLVL317 & LVL735 PE-PilA Fusion Protein

The ability of PE in the purified LVL317 PE-PilA fusion proteinconstruct to bind to vitronectin was evaluated. Microtiter plates(POLYSORP™, Nunc, Thermo Fisher Scientific) were coated with PE (fromvector pRIT16762) or with purified LVL317 PE-PilA fusion protein (10μg/ml). Plates were washed four times with NaCl 150 mM-polysorbate 20,0.05% (for example, TWEEN™ 20) and blocked for one to two hours withPBS-BSA 1%. After four washings, vitronectin (Vitronectin from humanplasma, SIGMA-ALDRICH®) was added (10 μg/ml), two fold diluted (12dilutions), and the plates were incubated for 1 h at room temperature.The plates were then washed 4 times with NaCl 150 mM-polysorbate 20,0.05% (for example TWEEN™ 20) After washings, the bound vitronectin wasdetected using peroxydase sheep anti-human vitronectin (US Biological)followed by the addition of ortho-phenylene diamine/H₂O₂ substrate. Thecolor developed is directly proportional to the amount of antibody fixedto the vitronectin.

See FIG. 23 for (a) LVL317 PE-PilA fusion protein bound to vitronectin.PilA=PilA from NTHi strain 86-028NP (as described for pRIT16790);PE=Protein E (as described for pRIT16762) and (b) LVL317 and LVL735PE-PilA fusion protein bound to vitronectin.

Example 17: Vibronectin Binding. Inhibition of Vibronectin Binding byAntibodies Directed Against the LVL291 PE-PilA Fusion Protein

Microtiter plates (POLYSORP™, Nunc, Thermo Fisher Scientific) werecoated with PE (from vector pRIT16762) or with purified PE-PilA fusionprotein (10 μg/ml). Plates were washed four times with NaCl 150mM-polysorbate 20, 0.05% (for example, TWEEN™ 20) and blocked for twohours with PBS-BSA 1%. After washings, vitronectin (Vitronectin fromhuman plasma, SIGMA-ALDRICH®) was added at 50 μg/ml and purifiedantibodies anti-PE-PilA (produced and purified in house) were two-foldserially diluted and incubated for 1 h at room temperature. The plateswere then washed 4 times with NaCl 150 mM-polysorbate 20, 0.05% (forexample, TWEEN™ 20). After four washings, the bound vitronectin wasdetected using peroxydase sheep anti-Vitronectin (US Biological)followed by the addition of ortho-phenylene diamine/H₂O₂ substrate. Thecolor developed is directly proportional to the amount of antibody fixedto the vitronectin.

Inhibition of vitronectin binding to PE by polyclonal antibodiesdirected against PE-PilA was observed.

See FIG. 24 for inhibition of vitronectin binding by polyclonalantibodies against PE-PilA fusion protein.

Example 18: Antigenicity of LVL291 PE-PilA Fusion Protein. ELISA

Purified LVL291 PE-PilA fusion protein was validated in an antigenicitytest with monovalent proteins as control. The fusion protein was testedin a sandwich ELISA developed with polyclonal antibodies (rabbit andguinea pig) generated against the PE gene fragment coding for aminoacids 22 to 160 of SEQ ID NO: 4 (as described for pRIT16711) or againstPilA from NTHi strain 86-028NP (from vector pRIT16790).

PilA or PE was added at 100 ng/ml and serially two fold diluted. After30 minutes incubation and after washing, the bound antigen was detectedby a rabbit polyclonal serum obtained after immunisation with PE orPilA. The bound antibodies were detected using a peroxydase anti-rabbitIg (Jackson ImmunoResearch Laboratories, Inc.) followed by the additionof ortho-phenylene-diamine/H₂O₂ substrate. The color developed isdirectly proportional to the amount of antigen present. Absorbancereadings were measured using a spectrophotometer for microtiter plates.The antigenicity of the samples was determined by comparison to thecurve of the full length PE or full length PilA reference antigen and isexpressed in ug/ml. The reference represented 100% of antigenicity.

As observed in the Table 6: Antigenicity was observed with the purifiedLVL291 PE-PilA fusion protein compared to the monovalent PE and PilAantigens.

TABLE 6 Relative antigenicity obtained with purified LVL291 PE-PilAfusion protein in the antigenicity test. PE relative antigenicity (%)Protein E as Reference 100 PE-PilA 130-148 PilA as Reference 100 PE-PilA120-152

Example 19: Immunogenicity of LVL735 PE-PilA Fusion Protein

Female Balb/c mice (n=34) were immunized by the intramuscular route atdays 0, 14 and 28 with 50 μl of vaccine formulation containing 1, 0.2 or0.04 μg of PE-PilA fusion protein LVL317 or LVL735 formulated withinAS01_(E) or AlPO₄ (aluminium phosphate). The antibody responses to PEand PilA were determined in individual sera collected at day 42 and theIgG level against PE and PilA was measured and expressed in μg/ml.

See FIG. 27 for PE and PilA antibody response to LVL317 and LVL735.GMC=geometric mean concentration. GMT=geometric means titer.IC=confidence intervals.

Example 20: Protective Efficacy of the LVL735 and LVL317 Fusion Proteinsin a Mouse Model of Non-Typeable Haemophilus influenzae NasopharyngealColonization

Female Balb/c mice were intranasally immunized at days 0 and 14 with 10μl of vaccine formulation containing 5.8 μg of LVL735 or LVL317 admixedwith 0.5 μg of E. coli labile toxin (LT). A booster dose of 5.8 μg ofnon-adjuvanted LVL735 or LVL317 was administered at day 28. Control micewere vaccinated with LT alone at days 0 and 14, and PBS at day 28.Animals were intranasally challenged with 5×10⁶ cfu of NTHi 3224A strainat day 42. Bacterial colonies were counted in nasal cavities removed 1and 2 days after the challenge (n=10/time-point). Nasal cavities arehomogenized in medium and a bacterial quantification is performed.Results are well expressed in CFU/ml.

See FIG. 28 for the effect of LVL735 and LVL317 vaccination on bacterialclearance in a mouse model of non-typeable Haemophilus influenzaenasopharyngeal colonization.

The invention claimed is:
 1. A method for the treatment of otitis mediacaused wholly or in part by non-typeable Haemophilus influenzae in asubject in need thereof comprising administering to said subject atherapeutically effective amount of an immunogenic compositioncomprising the fusion protein of SEQ ID NO. 177 (QIQKAEQN DVKLAPPTDVRSGYIRLVKN VNYYIDSESI WVDNQEPQIV HFDAVVNLDK GLYVYPEPKR YARSVRQYKILNCANYHLTQ VRTDFYDEFW GQGLRAAPKK QKKHTLSLTP DTTLYNAAQI ICANYGEAFSVDKKGGTKKA AVSELLQASA PYKADVELCV YSTNETTNCT GGKNGIAADI TTAKGYVKSVTTSNGAITVK GDGTLANMEY ILQATGNAAT GVTWTTTCKG TDASLFPANF CGSVTQ).
 2. Amethod for the treatment of otitis media caused wholly or in part bynon-typeable Haemophilus influenzae in a subject in need thereofcomprising administering to said subject a therapeutically effectiveamount of an immunogenic composition comprising the fusion protein ofSEQ ID NO. 219 (IQKAEQND VKLAPPTDVR SGYIRLVKNV NYYIDSESIW VDNQEPQIVHFDAVVNLDKG LYVYPEPKRY ARSVRQYKIL NCANYHLTQV RTDFYDEFWG QGLRAAPKKQKKHTLSLTPD TTLYNAAQII CANYGEAFSV DKKGGTKKAA VSELLQASAP YKADVELCVYSTNETTNCTG GKNGIAADIT TAKGYVKSVT TSNGAITVKG DGTLANMEYI LQATGNAATGVTWTTTCKGT DASLFPANFC GSVTQ).
 3. A method for the treatment of a H.influenzae infection or disease in a subject in need thereof, saidmethod comprising administering to said subject a therapeuticallyeffective amount of an immunogenic composition comprising the fusionprotein of SEQ ID NO. 177 (QIQKAEQN DVKLAPPTDV RSGYIRLVKN VNYYIDSESIWVDNQEPQIV HFDAVVNLDK GLYVYPEPKR YARSVRQYKI LNCANYHLTQ VRTDFYDEFWGQGLRAAPKK QKKHTLSLTP DTTLYNAAQI ICANYGEAFS VDKKGGTKKA AVSELLQASAPYKADVELCV YSTNETTNCT GGKNGIAADI TTAKGYVKSV TTSNGAITVK GDGTLANMEYILQATGNAAT GVTWTTTCKG TDASLFPANF CGSVTQ).
 4. A method for the treatmentof a H. influenzae infection or disease in a subject in need thereof,said method comprising administering to said subject a therapeuticallyeffective amount of an immunogenic composition comprising the fusionprotein of SEQ ID NO. 219 (IQKAEQND VKLAPPTDVR SGYIRLVKNV NYYIDSESIWVDNQEPQIVH FDAVVNLDKG LYVYPEPKRY ARSVRQYKIL NCANYHLTQV RTDFYDEFWGQGLRAAPKKQ KKHTLSLTPD TTLYNAAQII CANYGEAFSV DKKGGTKKAA VSELLQASAPYKADVELCVY STNETTNCTG GKNGIAADIT TAKGYVKSVT TSNGAITVKG DGTLANMEYILQATGNAATG VTWTTTCKGT DASLFPANFC GSVTQ).
 5. The method of claim 4,wherein the H. influenzae infection or disease is a non-typeableHaemophilus influenzae infection or disease.